Treatment of Leydig cells with 1 mM 8-Br-cAMP for 48 h decreased microsomal cytochrome P-450 activities, 17 alpha-hydroxylase and C17-20 lyase, by 60-75% and resulted in desensitization of the steroidogenic response. Reduction of the oxygen tension from 19 to 1% O2 prevented the decrease in P-450 activities but not the reduction in steroidogenic capacity. The decrease in activity was also prevented by blocking steroid synthesis with aminoglutethimide. Treatment of cultures with steroid products, androstenedione or testosterone, or product analogs, epitestosterone or 17 alpha-methyltestosterone, at a concentration of 2 microM, which is equivalent to the concentration of testosterone resulting from stimulation with cAMP, also caused oxygen tension-sensitive decreases in hydroxylase activity. Treatment of cultures with 2 microM cortisol, estradiol, or methyltrienolone, an androgen receptor agonist, did not decrease hydroxylase activity, nor did treatment with an androgen receptor antagonist prevent the cAMP- or testosterone-induced decreases in hydroxylase activity. Reductions in hydroxylase and lyase activities resulting from testosterone- or epitestosterone-treatment had little or no effect on acute cAMP-stimulated testosterone production, whereas desensitization with cAMP caused an 80-90% reduction in steroidogenic capacity. 22R-Hydroxycholesterol-supported testosterone synthesis was decreased by both cAMP- and steroid-treatment at 19% O2, but not below the cAMP-stimulated level. Reduction of the oxygen tension partially prevented this decrease. These data are consistent with the hypothesis that the decline in microsomal P-450 enzymes in desensitized Leydig cells results from product (pseudosubstrate)-induced, oxygen-derived, free-radical damage rather than a steroid receptor-mediated process.