The direct effects of GnRH and its agonistic and antagonistic analogs upon testicular androgen biosynthesis were studied in primary cultures of testicular cells obtained from adult hypophysectomized rats. Treatment of cultured cells with hCG (10 ng/ml) substantially increased testosterone production, while concomitant addition of GnRH or its agonist [des-Gly10, D-Ser(TBu)6,Pro9NHEt-GnRH] decreased hCG-stimulated testosterone production in a dose-related manner with ED50 values of 1.2 X 10(-9) and 4.5 X 10(-11) M, respectively. Treatment with 10(-6) M of either a GnRH partial peptide or a cyclic GnRH analog did not affect hCG action; however, the addition of a GnRH antagonist ([Ac-D-Phe1,D-p-Cl-Phe2,D-Trp3,6]GnRH) together with hCG and GnRH blocked the GnRH-induced decrease in testosterone production, with a half-maximal inhibitory dose ratio (antagonist to GnRH) of 0.15. The stimulatory effect of hCG became apparent by 8 h of incubation; no hCG effect was seen at this time in the presence of GnRH. Treatment with hCG increased cAMP accumulation, but GnRH administration did not affect hCG-induced cAMP accumulation. In contrast, treatment with GnRH depressed testosterone production induced by cholera toxin or (Bu)2cAMP. The inhibitory effect of GnRH on testosterone production (93% inhibition) was associated with decreases in hCG-induced 17 alpha-hydroxyprogesterone (39%) and delta 4-androstenedione (82%), but was not accompanied by a decrease in progesterone production. In cells incubated with cyanoketone and spironolactone to prevent pregnenolone metabolism, hCG stimulated pregnenolone biosynthesis, while concomitant GnRH treatment did not affect hCG action. In contrast, GnRH decreased hCG-induced testosterone production in cells treated with 10(-5) M progesterone. Similarly, GnRH decreased hCG-induced testosterone and androstenedione production in cells incubated with 10(-5) M 17 alpha-hydroxyprogesterone. The present results demonstrate that GnRH and its analogs exert direct actions on testicular cells through stereospecific recognition sites. The inhibitory effect of GnRH on testicular androgen production occurs at sites distal to the formation of cAMP and pregnenolone and may be due to decreases in the activity of the enzymes 17 alpha-hydroxylase and 17-20 desmolase.