A new carboxylesterase isozyme (EC 3.1.1.1), designated ES-30, is described in mouse liver. Two phenotypes were distinguished, ES-30A, a possible null type, was found in SPE/Pas and in other lines derived from Mus spretus, and ES-30B was found in BALB/cJ and other laboratory inbred strains. ES-30B is characterized by a distinct electrophoretic band when stained using 5-bromoindoxyl acetate as the substrate. After isolation and purification from other esterases by ion-exchange chromatography and molecular sieving, the molecular mass was estimated by two independent methods to be 62 and 64 kDa, respectively. The activity of ES-30B is higher in adult males than in females and can be stimulated in vivo by testosterone. The distribution of phenotypes on the progeny of a backcross series suggests a separate locus, Es-30, with the allele a for absence and b for presence of the isozyme. Locus Es-30 is shown to be closely linked to Es-2 and to Es-7 of cluster-2 on chromosome 8. The gene order Es-9--Got-2--(Es-2, Es-7, Es-30) is suggested.