AbstractIn the present paper, a study on the stereoselectivity of 25 lipases of animal and microbial origin towards homogeneous prochiral triglycerides is presented. All the lipases tested catalyse the hydrolysis of the chemically alike but sterically nonequivalent ester groups in trioctanoin and triolein with different degrees of stereobias, depending on the fatty acyl chain length of the substrate (Rogalska et al., J. Biol. Chem. 256:20271–20276, 1990). Hydrolysis of thesn‐2 ester group is catalysed by very few lipases and onlyCandida antarcticaA shows a clear preference for this position. Most of the lipases investigated (12 with trioctanoin and 16 with triolein) showed a preference for thesn‐1 position. Using trioctanoin as substrate we observed a total stereoselectivity for positionsn‐1 withPseudomonassp. andPseudomonas aeruginosaand for positionsn‐3 withCandida antarcticaB. This was not the case with triolein as substrate. Among the 23 lipases studied here and the other two lipases described previously (Rogalska et al., J. Biol. Chem. 256:20271–20276, 1990), 17 show a higher stereoselectivity with trioctanoin than with triolein. With guinea pig pancreatic lipase and with three mold lipases (Geotrichum candidumM,Geotrichum candidumA, andCandida antarcticaB), the preference switches fromsn‐3 tosn‐1 when the acyl chain length increases from eight to 18 carbon atoms. The main conclusion to emerge from the present study is that the specific stereopreference of each lipase for a given substrate under given lipolytic conditions can be said to be its fingerprint. © 1993