We have previously demonstrated that cyclosporine inhibits testosterone (T) biosynthesis in vivo. To better understand the mechanism by which CsA inhibits T synthesis, interstitial cells were isolated from rat testes and incubated in the standard medium 199 with or without CsA (0-10 micrograms/ml) in the presence or absence of human chorionic gonadotropin (hCG, 10(-7) M) and 8-bromo cyclic AMP (cAMP, 0.5 mM) for 3 hr at 32 degrees C. The levels of cAMP and T were determined by RIA. CsA did not inhibit the basal secretion of T, but inhibited hCG-stimulated T production in a dose-dependent manner (4 ng/10(6) cells vs. 10 ng/10(6) cells at a CsA dose of 5 micrograms/ml, P less than 0.05). Radioligand binding of 125I-hLH to testicular membranes was not affected by CsA, as CsA did not compete with hCG/LH for binding sites (25-28% binding with or without CsA). Similarly, the MIX-stimulated cAMP production was not affected by CsA (24.03 +/- 1.09 vs. 20.60 +/- 0.38 pmol/10(6) cells), suggesting that CsA does not inhibit the accumulation of the second messenger. However, when interstitial cells were incubated with CsA in the presence of cAMP, a significant dose-dependent decline in T secretion was observed (7 ng/10(6) cells vs. 20 ng/10(6) cells at a CsA dose of 5 micrograms/ml). To determine whether CsA inhibits the steps beyond cAMP stimulation of T secretion, the kinetic parameters (Km and Vmax) of steroidogenic enzymes, delta 4-3 keto-17 alpha hydroxylase (17 alpha-hydroxylase), and delta 4-3 keto-17 beta hydroxy steroid dehydrogenase (17B-HSD) were determined by using Michaelis Menten analysis. Results are shown in the presence of CsA vs. no CsA: Km and Vmax values for 17 alpha-hydroxylase were (2.32 vs. 7.98 microM) and (27.96 vs. 100.97 pmol/mg protein/min), respectively. For 17B-HSD the Km and Vmax were (2.14 vs. 1.52 microM) and (15 vs. 15 pmol/mg protein/min), respectively. These results indicate that CsA inhibits the activity of 17 alpha-hydroxylase uncompetitively and 17B-HSD activity competitively. In conclusion the primary site for CsA inhibition is the cAMP stimulation and, CsA inhibits T synthesis at multiple sites.