Cytochrome P450c17 is a single enzyme that catalyzes two successive reactions within the delta 5 and delta 4 pathways. The proteins expressed with human, bovine, and rat cDNAs convert both pregnenolone and progesterone into delta 5-delta 4-C19 steroids, although the rat cDNA prefers the delta 4 pathway. Our results showed that the guinea pig adrenal possesses the enzymatic machinery to produce C19 steroids and suggest that the lyase activity plays a major role in regulating these syntheses. To obtain more information on the structure-function relationship we isolated a full-length cDNA clone encoding guinea pig P450c17. Northern blots of total RNA extracted from the testis, ovary, and adrenals of the guinea pig show that the P450c17 cDNA hybridized with a predicted 1.8-kb mRNA and with two other mRNAs of 3 and 4 kb. No signal other than the 1.8-kb mRNA was observed in the human adrenocortical NCI-H295 cells. Activation of the cAMP-dependent protein kinase A pathway increased the levels of the three mRNAs. Transfection of vectors expressing guinea pig P450c17 cDNA into nonsteroidogenic cells confers 17 alpha-hydroxylase and 17,20-lyase activities, showing that a single protein in the guinea pig supports both activities. However, the analysis of the enzymatic properties showed that the guinea pig P450c17 recombinant, in contrast to the human, supports hydroxylase and lyase activities only with delta 4 substrates. These results were further confirmed with isolated guinea pig adrenocortical cells. Our data demonstrate, first, that guinea pig P450c17 cDNA hybridizes with three different transcripts and second, that the expressed protein has characteristics associated exclusively with the guinea pig enzyme.