The deacylation reaction of diacetoxyscirpenol (DAS), a trichothecene mycotoxin, was studied by using five carboxylesterase isoenzymes isolated from mouse liver microsomes. The simultaneous isolation of the five microsomal carboxylesterase isoenzymes was accomplished by chromatofocusing. DAS serves as a unique substrate since both acetyl groups are accessible for hydrolysis, providing an opportunity to investigate the regioselective hydrolysis of DAS with the various isoenzymes. A novel basic carboxylesterase isoenzyme, pI 8.4-8.2, was isolated. This isoenzyme exhibits strict regioselectivity toward DAS hydrolysis; only the acetyl group at C-4, but not that at C-15, is hydrolyzed. Moreover, this is the major isoenzyme responsible for the deacylation of DAS in mouse hepatic microsomes. The rate constant determined for deacylation of DAS at C-4 for this isoenzyme is 130-1000 times greater than that of the other carboxylesterases. This isoenzyme was also the most efficient for the hydrolysis of 4-monoacetoxyscirpenediol with a rate constant 30-100 times greater than that of the other isoenzymes.