An intracellular carboxylesterase from Bacillus coagulans NCIMB 9365 with high stereospecific hydrolytic activity toward racemic esters of 1,2-O-isopropylideneglycerol was purified and characterized. The microorganism contained two intracellular carboxylesterases designated as carboxylesterase A and B. Purification was achieved in three steps : precipitation with ammonium sulfate from the crude cellular lysate, ion exchange, and gel filtration chromatography. Carboxylesterase A is stereoselective, has a molecular weight of 70-73 kDa with an isoelectric point of 4.7, and a maximum activity (assayed on alpha-naphthylcaprylate) at pH 7.0 and 65 degrees C. The enzyme showed good affinity coward (R)-benzoyl-1,2-isopropylideneglycerol (K-m = 1.05 mM) and its activity on this substrate was competitively inhibited by (S)-benzoyl-1,2-isopropylideneglycerol. The purified enzyme yielded (A)-1,2-O-isopropylideneglycerol with high enantiomeric excesses (e.e.) by resolution of various racemic esters (97% e.e starting from benzoate ester).