A thermally stable esterase (SNSM-87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure-enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)-2-chloromandelates as the model substrates, an expanded Michaelis-Menten mechanism for the rate-limiting acylation step is adopted for the kinetic analysis. The Bronsted slope of 25.7 for the fast-reacting (S)2-chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow-reacting (R)-2-chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is rate-limiting. However, the rate-limiting step shifts to the formation of tetrahedral intermediates for the (S)-2-chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate.