The substrate specificity of rat testicular and epididymal peptidase was investigated using chromogenic substrates, D. L-alanine-, L-arginine-, gamma-N-L-glutamine-, L-leucine-, D. L-methionine-, alpha-N-benzoyl-D. L-arginine-, and N-benzoyl-L-leucine-beta-naphthylamide. The histochemical distribution of peptidase activity demonstrated with these substrates was also investigated in the testis and epididymis. L-Arginine-beta-raphthylamide (Arg-beta-NA) and gamma-N-L-glutamine-beta-naphthylamide (Glu-beta-NA) were mostly hydrolyzed in the testis and epididymis, respectively. Histologically, the activity using Arg-beta-NA as substrate (aminopeptidase B) appeared in both the cytoplasms and nuclei of interstitial cells and spermatogonia and the heads of spermatozoa, while activity using other substrates was found only in the cytoplasms of cells in the germinal epithelium. In the epididymis, strong reaction with Glu-beta-NA (gamma-glutamyl transpeptidase) was found in the apical part of the epithelial cells and in the heads of spermatozoa. Neither alpha-N-benzoyl-L-arginine- nor N-benzoyl-L-leucine-beta-naphthylamide was utilized in either the testis or the epididymis.