In vivo and in vitro experiments were designed to assess the effect of testosterone on aldosterone secretion in male rats. Orchidectomized rats were injected subcutaneously with oil or testosterone propionate ([TP] 2 mg/kg) for 7 days. Intact rats were injected with oil only. The results indicate that the plasma aldosterone level was higher in orchidectomized versus intact and TP-replaced rats. In the in vitro study, testosterone caused a marked decrease of aldosterone secretion by zona glomerulosa (ZG) cells, but failed to alter the accumulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Testosterone significantly decreased the corticotropin (ACTH)-stimulated production of aldosterone and accumulation of cAMP in rat ZG cells. The conversion of corticosterone to aldosterone and of 25-OH-cholesterol to pregnenolone, as well as angiotensin II (ANG II)-stimulated production of aldosterone, were decreased by testosterone. These results suggest that testosterone inhibits the basal and ANG II- and ACTH-stimulated release of aldosterone, via inhibition of aldosterone synthase activity and cytochrome P-450 side-chain cleavage (P450scc) activity, and ACTH-stimulated cAMP accumulation in rat ZG cells.