AbstractProduction of testosterone by highly purified Leydig cells prepared from rat and mouse testes is compared. Testosterone formation is improved to a higher degree in rat (2.7‐fold) than in mouse (1.7‐fold) cells by callagenase treatment of the testis compared with mechanical isolation. Mouse Leydig cells respond to exogenous stimuli (chorigonadotropin, dibutyryl cyclic AMP) with 2.4‐fold higher testosterone secretion than rat cells. A 1.7‐fold increased conversion of androgen precursors to testosterone by mouse compared with rat Leydig cells is demonstrated in static incubations as well as in steady‐state superfusion experiments and can be derived from enhanced androstenedione reduction and a less inhibitory effect of progesterone on this process is mouse Ley