Decapsulated testes from adult rats were digested with collagenase, and the fraction enriched in germinal and Leydig cells was applied to a 0-4% continuous metrizamide gradient and centrifuged. This leads to separation of a germinal cell fraction and two putative Leydig cell populations that bind human choriogonadotropin, but only one of which responds to the gonadotropin with marked increase in testosterone production. Adenylate cyclase activity was present in these three fractions, and Mn2+ was more effective than Mg2+ as a divalent cation. The adenylate cyclase activity associated with the germinal cell fraction was just marginally stimulated by fluoride and by the non-hydrolyzable GTP analog 5'-guanylimidodiphosphate, while that associated with the Leydig cell populations was stimulated to a greater degree depending upon the type of divalent cation. Only the Leydig cell populations exhibited marked human choriogonadotropin-sensitive stimulation of adenylate cyclase activity in the presence of 5'-guanylimidodiphosphate above that observed with the GTP analog alone. These results suggest the presence of distinct adenylate cyclases in adult rat testis and indicate that both populations of Leydig cells are capable of producing cyclic AMP in response to gonadotropins such as human choriogonadotropin.