Esterase-9A, which appears electrophoretically as a triplet of the bands III-50, III-40 and III-30, was isolated from the kidneys of male NMRI-mice by isoelectrofocusing and refocusing followed by repeated molecular sieve chromography. The overall purification was approx. 250 fold and each of the three bands was isolated separately. The band of the triplet nearest to the cathode, III-50, changed in vitro into the satellite bands III-40 and III-30 and, further, into the band III-22 not observed before in the homogenate. It is assumed that the band III-50 represents the original gene product. The molecular weight (45 000) of the band III-50 is identical with those of III-40 and III-30, as measured by analytical electrophoresis, whereas the molecular weight obtained by thin-layer chromatography was 51 000. There were no obvious signs that esterase-9 was composed of subunits. The Km constant for 4-nitrophenyl proprionate was identical for each of three bands. The esterase-9A is the first testosterone-dependent isozyme of the mouse carboxylesterase (carboxylicester hydrolase, EC 3.1.1.1) system which has been isolated.