Isolation of a novel microbial lipase (EC 3.1.1.3) having specific catalytic activity for the synthesis of optically pure 2-O-benzylglycerol-1-acetate, the building block for the preparation of many beta-blockers, phospholipase A2 inhibitors and other biologically active compounds was the aim of this investigation. A Pseudomonas (strain G6), recently isolated from soil, produced an extracellular lipase. SDS-PAGE analysis showed that the lipase protein was a hexamer. The molecular weight of the sub-units of the lipase protein were 10, 19, 29, 30, 47 and 53. The catalytic activity of the lipase was exploited for the synthesis of 2-O-benzylglycerol-1-acetate from 2-O-benzylglycerol through transesterification using vinyl acetate as acylating agent. High selectivity of the lipase towards the monoacetate product was demonstrated. A 97% enantiomeric excess (ee) of S(+)-2-O-benzylglycerol-1-acetate was obtained when the reaction was carried out at room temperature with shaking. The lipase was highly active in anhydrous organic microenvironments and in non-polar organic solvents with log P values above 2.5.