In this work, the lipase (Pseudomonas cepacia) (PS)- and Candida rugosa lipase (CRL)-catalyzed asymmetric deacylations of the esters of structurally different secondary alcohols were studied. Enantioselectivity in the case of lipase PS was always easily predicted according to the sizes of the substituents at the alcoholic stereocenter whereas the favored enantiomer in the case of CRL strongly depended on the structure of the racemic alcohol moiety in an ester. In the next step, commercial CRL was fractionated into its isoenzymes (A and B). The two isoenzymes A and B always gave the same enantiopreferences. This observation clearly showed that the heterogeneity of the commercial CRL was not a reason for the reversed enantioselectivity of certain substrates such as cyanohydrin, ol-hydroxycarboxylic acid, and 2,2,2-trihaloethanol derivatives when compared to the selectivity observed when lipase PS was used as a catalyst.