The testes of testicular feminized (Tfm) mice synthesize and secrete abnormally low amounts of testosterone, as a consequence of selectively decreased cytochrome P450(17 alpha) activity. To investigate the mechanism of this deficiency, three steroidogenic enzymes were immunolabeled in the testes of normal and Tfm adult (2.5-6 month old) mice. Cholesterol side-chain cleavage cytochrome P450 (P450scc) and delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were detected in the Leydig cells of both normal and Tfm mice whereas, in contrast to normal mice, only a small proportion of Leydig cells were immunostained for cytochrome P450-17 alpha-hydroxylase, C17-->20 lyase (P450(17 alpha)) in the testes of Tfm mice. The numbers of cells differed from male to male and interestingly were markedly higher in the right testis. Explants of testes from Tfm mice were kept in organ culture at 32 degrees C for 45 h, with or without dibutyryl cyclic AMP (100 or 500 mumol/l). All Leydig cells remained positive for P450scc and 3 beta-HSD, and P450(17 alpha) became detectable in the majority of Leydig cells in both left and right testes, showing that the lack of expression of P450(17 alpha) protein in Tfm mouse testes in vivo is not structural but is a regulatory phenomenon.