A collection of salt-sensitive vacuolar (ssv) Saccharomyces cerevisiae mutants were selected for analysis in an attempt to reveal components of the osmosensing signal transduction pathway. A previous screen of these mutants had been used to select those with an impaired glycerol response to salt stress. In this study the glycerol-3-phosphate dehydrogenase activity was measured in all the strains which showed reduced glycerol accumulation to demonstrate a corresponding low enzyme activity which could be caused by lack of signalling through the high osmolarity glycerol (HOG) response pathway. However, enzyme activity was found to be impaired in only one of the strains tested. This demonstrated that the measurement of glycerol accumulation is not a particularly useful screening method for defects in the HOG pathway. The activity of the promoter of CTTl, another stress-responsive gene, was measured in selected ssv strains using a lacZ reporter gene attached to the CTTl promoter stress response element. This gave further information about the stress-responsiveness in the strains tested. CTTl promoter activity did not correlate with GPDH activity in all of the strains tested. As CTTl is subject to control by more than one type of stress the results imply that in at least one of the strains another stress response may be impaired. The VACl homologue (VACIH) on chromosome XIV was characterised as a candidate for one of the 55V genes, 55W7. Although it was demonstrated not to be 55W 7, a role for VACIH in vacuolar protein sorting was discovered. The Δvaclh strain also displayed class E vacuolar morphology. Sequence analysis and complementation experiments demonstrated that VACIH is identical to VPS27.