Nucleophilic properties of amino-acids were studied systematically in acyl-transfer reactions catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal- l-Ala- l-Ala- l-PheOMe as the acyl-group donor. In α-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: d-AlaNH 2 < GlyNH 2 < l-AlaNH 2 < l-ScrNH 2 < l-ThrNH 2 < l-HisNH 2 < l-ValNH 2 < l-LcuNH 2 < l-TrpNH 2 < l-MetNH 2 < l-NvaNH 2 < l-PheNH 2 < l-IleNH 2 < l-TyrNH 2 < l-ArgNH 2 . In reactions catalyzed by subtilisin 72, the reactivities increase as follows: l-LeuNH 2 < l- IleNH 2 < l-ThrNH 2 < l-ArgNH 2 < l-TrpNH 2 < l-NvaNH 2 < l-ValNH 2 < l-MctNH 2 < l-AlaNH 2 < l-ScrNH 2 < d-AlaNH 2 < GlyNH 2 . In α-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of d-AlaNH 2, l-ArgNH 2 and l-TyrNH 2. In the reactions catalyzed by subtilisin 72, Amino-acid side-chain characteristics and the nucleophile reactivities are not related. The data obtained show the low selectivity of the S 1′ subsite of subtilisin 72 and high specify of this subsite in α-chymotrypsin.