Immunocytochemical studies and mRNA measurements have shown that the rat epididymis--like the rat prostate--expresses both rat steroid 5alpha-reductase isozymes, i.e. type I and II. So far, enzyme activity measurements in rat epididymis homogenates, however, do not support the presence of type I 5alpha-reductase activity. Incubating homogenates of both tissues with a wide range of substrate concentrations, we were able to detect activity of both isozymes in rat prostate and epididymis tissues at neutral pH. In rat prostate the amount of type I activity, as measured by the Vmax at pH 7.0, exceeds that of type II 5alpha-reductase 50-fold. The efficiency ratio, Vmax/Km, of the type I isozyme accounts for 25% of the total in vivo potential activity. A possible anabolic role for the type I isozyme in rat prostate was thus surmised. In rat epididymis the Vmax of type I and type II 5alpha-reductase at pH 7.0 were similar. Comparison of the efficiency ratio Vmax/Km of either isozyme in the rat epididymis, however, suggested that the type II isozyme would play the major role in the 5alpha-reduction of testosterone at physiological concentrations and at neutral pH. The specific localization of the isozymes should be considered to allow for correct quantification of their in vivo contribution to dihydrotestosterone formation.