Enzymatically dispersed testis cells derived from 7-day-old male rats maintained their gonadotropin-stimulated testosterone production for 18 days in culture. Treatment with hCG or LH stimulated androgen production in a dose-dependent manner, with ED50 values of 0.030 +/- 0.007 and 1.0 +/- 0.4 ng/ml for hCG and LH, respectively. Concomitant treatment with a phosphodiesterase inhibitor further enhanced LH action. In contrast, treatment with FSH, GH, or PRL was without effect. Treatment with forskolin, cholera toxin, or 8-bromo-cAMP induced dose-dependent increases in testosterone biosynthesis; this was accompanied by stimulation of 3 beta-hydroxysteroid dehydrogenase activity after treatment with hCG, forskolin, or 8-bromo-cAMP. RIA measurement of different androgens in HPLC-fractionated medium revealed that the main androgen secreted by the neonatal testis cells was testosterone, with lower production of 5 alpha-androstane-3 alpha,17 beta-diol and negligible 5 alpha-dihydrotestosterone, androstenedione, and androsterone. Treatment with epidermal growth factor, GnRH, and arginine vasopressin (AVP) decreased hCG-induced testosterone biosynthesis. Since the inhibitory actions of GnRH and AVP were blocked by concomitant addition of specific hormone antagonists, their inhibitory actions were probably mediated by specific testis receptors. In contrast, treatment with several potent synthetic steroid hormone analogs [diethylstilbestrol (an estrogen), dexamethasone (a glucocorticoid), R5020 (a progestin; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R1881 (an androgen; 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one), or cyproterone acetate (an antiandrogen; 17 alpha-acetyloxy-6-chloro-1,2-dihydro-(1 beta,2 beta)3'-H-cyclopropa-(1,2) pregna-1,4,6-trien-3,20-dione)] did not affect testosterone biosynthesis in hCG-treated cells. These results demonstrate that testosterone production by neonatal testis cells is maintained by gonadotropins during prolonged culture; the ability of cAMP-generating drugs and a cAMP analog to mimic gonadotropin actions on testosterone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity suggests a mediatory role of cAMP in gonadotropin action; and AVP, epidermal growth factor, and GnRH, through their putative testis receptors, directly inhibit gonadotropin-stimulated testosterone synthesis, while various steroids (androgens, estrogens, progestins, and glucocorticoids) do not affect Leydig cell function in the neonatal testis. The present culture system offers a unique model for elucidating the hormonal control of Leydig cell androgen biosynthesis during neonatal development.