Ketoconazole is a well-tolerated, synthetic, imidazole derivative currently in widespread therapeutic use against mycotic infections. Recent evidence that it depresses testosterone synthesis in humans prompted us to investigate the effects in rats of its administration alone or in combination with the gonadotropin releasing hormone superagonist analogue leuprolide. Plasma luteinizing hormone, testosterone, and ketoconazole levels as well as ventral prostate weight and tumor growth in rats bearing the androgen-dependent Dunning R3327H model of prostate adenocarcinoma were measured. Doses of 30 mg/kg twice daily of ketoconazole alone depressed plasma testosterone levels by approximately 75% to a nadir of 0.47 +/- 0.08 (SE) ng/ml on day 20 (P less than 0.001 versus basal). This effect of ketoconazole was exerted directly at the testicular level since plasma luteinizing hormone levels were not suppressed. In response, ventral prostate weight declined and growth of the Dunning R3327H tumor was retarded to rates observed in castrate controls. Leuprolide alone lowered basal testosterone levels to 0.20 +/- 0.02 ng/ml after 35 days of daily administration but persistent androgen increments after each injection (acute-on-chronic effect) were observed (i.e., to 4.41 +/- 0.62 ng/ml). The addition of ketoconazole to leuprolide inhibited the acute-on-chronic rise in testosterone to 0.33 +/- .07 ng/ml and also lowered basal testosterone levels further to 0.11 +/- 0.01 on day 10 of combined administration. Ketoconazole also blunted the response to the first injection of leuprolide from a 3-h peak level of 8.74 +/- 0.53 to 4.17 +/- 0.80 with the 40 mg/kg dose. These results indicate that combining ketoconazole with leuprolide achieves greater suppression of testosterone than either agent alone. When such protocols are applied to humans with prostate cancer, more extensive effects may be expected because of the greater sensitivity of patients than of the rodent species to these agents.