Racemic 2-acyl-3-hydroxymethykl-butanolides were resolved through a @se-catalyzed acylation with acetic anhydride. Optically active forms of 2-butyryl-3-hydroxymethyl-4-butanolide 14 obtained were used to prepare enantiomers of IM-2 10, the autoregulator from Srrepfomyces sp. FRI-5. The absolute configuration of IM-2 was deduced to be (2R,3R,6R). In Streptomyces, a number of butyrolactone autoregulators which control cytodifferentiation or secondary metabolites production are known, such as A-factor 1 I, factor-l 22, Grafe's factors 3, 4 and S3, virginiae butanolides (VB) A-E 3, 6, 7, 8 and 94, and IM-2 105. These molecules have a common 2,3disubstituted butanolide skeleton but differ in the C-2 side chain. All of autoregulators which have a C-6 hydroxyl group possess a 2,3-rrans configuration, but stereochemistry of a C-6 hydroxyl group is different among them as shown in Fig. 16. The absolute configurations of A-factor 1 and VBs (A, B and C 3,6 and 7) have been assigned to (3R) and (2R, 3R, 6S), respectively, with their chiral synthesis from (S)-paraconic acid being carried out by Mori et al. ', but those of other molecules are not determined as yet. Since autoregulators of Streptomyces are usually produced in trace amounts in culture broths, synthetic samples are very important to investigate their functions. 2-Acyl-3-hydroxymethyl-4-butanolides, one of which is Afactor 1, are synthetic intermediates of autoregulators with a C-6 hydroxyl group' and also biosynthetic precursors of them*. Recently, in order to study the substrate stereospecificity of the enzyme concerning the reduction from 3-hydtoxymethyl-2-(5-methylhexanoyl)-4-butanolide 11 to VB-A 3, we have attempted to prepare optically active forms of 11 by resolution of its racemate. As a result, enantiomers of 11 each having high optical purity have been obtained using a lipase-mediated acylation. In this paper, we describe the enzymatic resolution of 11 and its congeners, and its application to a preparation of enantiomers of IM-2 10, which led to determine the absolute configuration of 10 as (2R, 3R, 6R).