Modes of inhibition and binding of ketoconazole, an orally antimycotic agent, to NADPH-cytochrome P-450dependent enzymes were investigated using subcellular fractions of human and rat testes, human adrenocortical adenoma tissue and rat adrenals and livers. Ketoconazole competitively inhibited the activities of steroid17α-hydroxylase and C17-20lyase in rat and human testes, 16α-hydroxylase in human testes and 21-hydroxylase in rat adrenal glands. Ki values were in the order of10^-8M for human testicular enzymes, while the order was10^-7-10^-6M for rat adrenal and testicular enzymes. Kinetic studies indicated that ketoconazole bound to cytochrome P-450and not to other components of monooxygenase systems. Spectrophotometric studies also revealed direct binding of ketoconazole to cytochrome P-450 component by inducing type II difference spectra in all tissue preparations examined, indicating that ketoconazole is possibly a universal inhibitor of NADPH-cytochrome P-450dependent monooxygenases which are involved in metabolism of many substances including steroids, toxins, carcinogens and others.