The newly identified lipases of 67 bacterial strains, primarily Bacillus and Pseudomonas, from the ARS Culture Collection have been characterized on the basis of their positional specificity for triglycerides (triolein). Lipase was produced by growing the cultures in tryptone-glucose-yeast extract medium for 24 h at 30degreesC before addition of triglyceride. The lipase was allowed to act on the triglyceride for 3 days before analysis by thin-layer chromatography. Of the bacterial lipases tested, 55 displayed random specificity, 9 were 1,3-specific, and 3 showed no apparent lipase activity under these conditions.