AbstractA widely utilized pig liver esterase preparation has been found to be derived essentially exclusively from the cytosolic fraction of pig livers. Esterases in cytosol and microsomes prepared from a fresh pig liver hydrolyzed theS‐ andR‐enantiomers of racemic oxazepam 3‐acetate (rac‐OXA) with specific activity ratios of approximately 2.3:1 and 1:62, respectively. Product formations were analyzed by chiral stationary phase high‐performance liquid chromatography. The commercial pig liver esterase preparation showed greater activity towardS‐OXA than did the esterases in the cytosolic fraction prepared from fresh pig liver. The results established that (i) esterases contained in microsomes and cytosol of pig liver have opposite enantioselectivity in the hydrolysis ofrac‐OXA and (ii) the commercial pig liver esterase preparation has a cytosolic origin. © 1995 W