Flavomycin is a phosphoglycolipid antibiotic, which is used exclusively as a growth- promoting feed additive. Existing data, from both in vitro and in vivo ruminal studies, give conflicting results regarding its mode of action, as well as no clear microbiological basis for the observed responses. Studies however do indicate that the principal site of action of the antibiotic is the rumen. From the available data, flavomycin appears to promote growth in a manner distinct from that of other feed antibiotics, for which the growth-promoting mechanisms have been elucidated. This study aimed to characterise the effect that flavomycin has on the microflora of the rumen, allowing its growth promoting mechanism in ruminants to be determined. In vitro analysis demonstrated that flavomycin has only antibacterial activity, as ruminal species of protozoa, fungi and archaea were unaffected by the antibiotic. Of the ruminal bacterial species tested, Fusobacterium necrophorum, Fibrobacter spp. and certain hyper-ammonia producing (HAP) bacteria (Atopobium oviles, Desulfomonas sp. and Peptostreptococcus anaerobius) were highly sensitive to the antibiotic. The sensitivity of the Fibrobacter spp. to flavomycin suggested that flavomycin is likely to select for a cellulolytic bacterial flora comprised predominantly of Ruminococcus spp., as has been previously proposed on the basis of in vitro fermentation studies. In vivo, suppression of ruminal numbers of F. necrophorum and flavomycin sensitive HAP bacteria occurred as a result of flavomycin supplementation. It was demonstrated that these bacterial populations were highly variable, between individual sheep and days respectively, suggesting why previous studies produced conflicting results. Assessment of ruminal fermentation parameters demonstrated that flavomycin caused a significant decrease in the production of ruminal ammonia, which could be directly attributed to decreased numbers of ruminal HAP bacteria. A small increase in the ruminal concentration of lactate also occurred, which con-elated with the suppression of ruminal numbers of the lactate utilising F. necrophorum. No change in the balance of individual volatile fatty acids (VFA) occurred, however total VFA production was significantly decreased. This was likely to be due to the total viable anaerobic bacterial counts being lower during flavomycin supplementation, although this result was not statistically significant. Uncultured rumen bacteria were also implicated in the growth promoting mechanism of flavomycin. Molecular investigation of the rumen bacterial population by denaturing gradient gel electrophoresis (DGGE) demonstrated that several changes occurred, which correlated with flavomycin supplementation. Analysis of the sequence data obtained from excised DGGE bands highlighted that the majority of the operational taxonomic units (OTU) detected were represented by presently uncultured species of bacteria, of which almost half had not been previously identified. Identification of the flavomycin sensitive bacterial populations was not possible, however, due to the recovery of multiple sequences from individual DGGE bands. Existing bacterial 16S rDNA sequence data, from published ruminal clone libraries, also demonstrated the poor cultural representation of rumen bacterial diversity, with only 10% of the OTU detected being represented by cultured bacterial species. Based on these results, flavomycin has the ability to increase the efficiency of dietary protein utilisation, although the role of uncultured bacteria in the growth promoting mechanism of the antibiotic is not clear. Protein retention in the rumen is increased as a consequence of decreased deamination by ruminal HAP bacteria. F. necrophorum has the ability to attach to and damage rumen epithelium, as well as being the principal aetiological agent in the development of liver abscesses. Suppression of F. necrophorum is likely to decrease metabolic and immune burdens within the animal, as well as potentially reducing the rate of rumen wall tissue turnover. The use of flavomycin as a feed additive is to be banned in Europe in 2005. However, it is not known if presently available feed additives or treatments will be able to act as an effective replacement for this antibiotic. Characterisation of an adaptive resistance mechanism against flavomycin, in the ruminal bacterium Prevotella bryantii, demonstrated that cross-resistance to therapeutic antibiotics can occur. As a result of this finding, and the interest in development of phosphoglycolipid antibiotics for therapeutic application, it can be concluded that the withdrawal of the use of a flavomycin as a feed additive is a wise precautionary measure to ensure the long-term efficacy of this class of antibiotics.