Abstract The levels and subcellular distributions of various peptidase and esterase activities in a range of lactococcal and Streptococcus thermophilus strains were investigated. There was no correlation between the levels of the enzymes in the different strains and the ability of the strains to produce acid when grown in milk. While considerable differences between individual strains were apparent, average levels of X-prolyldipeptidyl aminopeptidase, dipeptidase and tripeptidase were similar in the Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris strains studied, while that of lysylaminopeptidase (i.e. activity assayed using lysine p-nitroanilide as substrate) in the L. lactis subsp. cremoris strains was approximately double that in the L. lactis subsp. lactis strains. The average levels of lysylaminopeptidase and X-prolyldipeptidyl aminopeptidase in the S. thermophilus strains studied were similar to those in the L. lactis subsp. cremoris strains, while the average levels of dipeptidase and tripeptidase were considerably lower. All peptidases studied were recovered predominantly in the cytoplasmic fraction, although in a few strains there was some evidence to suggest that a part of the tripeptidase activity may be associated with cell structures comprising the particulate fraction. The levels of esterase activity in the strains were considerably different between strains. However, the average level of esterase activity detected in the two lactococcal subspecies was similar, while that in the S. thermophilus strains was more than double the lactococcal average. The subcellular distribution of the esterase in all strains studied showed that a significant proportion of the activity is located on the cell surface.