The sizes of the large, medium, and small substituent recognition sites (L, M, and S pockets, respectively) in Candida rugosa lipase (CRL) were roughly estimated by measuring the specific hydrolytic activity against several p-nitrophenyl esters. These relative sizes were assessed as L pocket>phenyl, ethyl>M pocket>methyl>S pocket>hydrogen. The hydrolysis of a series of p-nitrophenyl esters of ω-substituted fatty acids was also examined. In this series, p-nitrophenyl esters having one methylene between the ester–carbonyl carbon and cyclohexyl, phenyl, or isopropyl moiety largely demonstrated decreases in hydrolytic activity.