Shiga toxin-producing Escherichia coli (STEC) has been recognized as an important foodborne pathogen. Of this group, O91 is one of the common serogroups frequently isolated from patients and food in some countries, with O91:H21 being previously implicated in hemolytic uremic syndrome (HUS). Cattle are principle reservoirs for STEC, and studies examining STEC shedding in cattle often include experimental inoculation of strains of interest using antibiotic resistance markers for identifiable recovery. However, indigenous fecal microbes exhibiting similar resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves, leading us to seek other, more effective, markers. Among our strategies was the development of a chromosomally integrated bioluminescence marker via transposon mutagenesis using a luxCDABE cassette from Photorhabdus luminescens and a plasmid borne GFP marker via transformation of the pGFP vector. The luxCDABE marker was inserted on host chromosome at a site that was 27 nucleotides before the stop codon of gene yihL and confirmed to have little impact on important virulence genes and growth rate with a very high stability. In contrast, plasmid borne GFP marker showed poor stability without the application of appropriate antibiotic selection pressure. For calves receiving luxCDABE-marked O91:H21, the fecal counts of the organismranged from 1.2 x 10 3 to 1.3 x 10 4CFU/g at two days post inoculation and decreased to 5.8 to 8.7 x 10 2 CFU/g or undetectable level after two weeks.Intestinal contents sampled from various positions at day 14 post inoculation indicated that cecum and descending colon may be the primary localization sites of this O91:H21 strain. Compared to antibiotic resistance markers, the use of bioluminescence markers does not require the restricted pre-inoculation screening of animals. The enumeration of luxCDABE-marked O91:H21 from feces and intestinal contents was easily accomplished and confirmed reliable by M-PCR analysis under the presence of indigenous bacteria which cannot be eliminated by antibiotic-supplemented selective plates. Therefore, the chromosomal integrated luxCDABE marker may be a better model for the study of STEC colonization and shedding in cattle.