The microsomal fraction from the testes of immature pigs can convert pregnenolone to 17-hydroxypregnenolone and also to 5,16-androstadien-3 beta-ol (andien-beta). The available evidence supports the hypothesis that both these reactions are catalysed by one enzyme, cytochrome-P450(17 alpha). In the absence of cytochrome b5, 17-hydroxypregnenolone will be the major product but that if cytochrome b5 is present in sufficient quantity, andien-beta becomes a major product. The point of divergence between the conversion of pregnenolone to either 17-hydroxypregnenolone or andien-beta was investigated using enzyme kinetic analysis to determine whether 16 alpha-hydroxypregnenolone, 20 beta-hydroxypregnenolone or 16-dehydropregnenolone could be specific intermediates to one reaction or the other. Product inhibition by 17-hydroxypregnenolone and andien-beta was competitive for both 17-hydroxylase and "andien-beta synthetase" supporting the current view of a common active site for both reactions. 16 alpha-Hydroxypregnenolone was a very poor competitive inhibitor of 17-hydroxylase and andien-beta synthetase with Ki(app) values many fold greater than the Km(app) for pregnenolone or the Ki(app) for reaction product, rendering it unlikely that 16 alpha hydroxylation is a key intermediary step in either pathway. 20 beta-Hydroxypregnenolone was a more potent inhibitor of andien-beta synthetase than of 17-hydroxylase and for the latter enzyme activity, the Ki(app) was lower than that for 17-hydroxypregnenolone itself. However, for andien-beta synthetase, 20 beta-hydroxypregnenolone may be an early intermediate as the Ki(app) was consistent with the affinity for the active site being intermediate between the Km(app) for pregnenolone and the Ki(app) for andien-beta. 16-Dehydropregnenolone was equipotent at inhibiting 17-hydroxylase and andien-beta synthetase activities suggesting that 16-dehydropregnenes may be involved in the stages immediately prior to C21 side-chain cleavage.