Ten different nonspecific esterases in both mouse (Mus musculus) and rat (Rattus norvegicus) testis were identified following the analysis of electrophoretic patterns using genetic, developmental, and biochemical criteria. None of the enzymes were unique to testis, although the pattern of activity was testis specific. The enzymes comprised, in each species, six carboxylesterases (EC 3.1.1.1), one arylesterase (EC 3.1.1.2), one acetylesterase (EC 3.1.1.6), and two butyrylesterases (tentative designation). Cholinesterase (EC 3.1.1.8) was not detected. Individual homology relationships were recognized between the two species for all of these activities, except three of the carboxylesterases; however, these were coded for by homologous gene clusters. Similarities between the two species extended to the developmental course of expression and the modulation of the pattern of activity by the testicular feminization (Tfm) mutation. We describe the effects of the sex reversal (Sxr) mutation in the mouse, as well as the distribution of individual activities between Leydig cells and seminiferous tubules. The results of earlier histochemical studies are interpreted in the light of the present investigation. The correspondence between mouse- and rat-testis esterases suggests that the results could serve as a basis for mammalian testis esterase systems in general.