Adrenarche is the prepubertal onset of increased adrenal secretion of 19-carbon steroids, especially dehydroepiandrosterone (DHEA). However, while human beings and chimpanzees exhibit adrenarche, other primates such as the baboon and rhesus monkey do not, and the adrenals of most other mammals produce little or no DHEA. Thus, the acquisition of adrenarche is a very recent evolutionary event. DHEA is produced from pregnenolone by the successive 17alpha-hydroxylase and 17,20 lyase activities of a single enzyme, P450c17. To ascertain whether sequence differences in P450c17 contribute to adrenarche, we cloned the rhesus monkey cDNA from adrenal tissue and cloned the chimpanzee and baboon cDNAs from genomic DNA using an exon-trapping strategy. Using microsomes from yeast transformed with rhesus, baboon, chimp, or human P450c17, we measured the Michaelis constant and maximum velocity for the 17alpha-hydroxylase and 17,20 lyase activities. The human and chimp enzymes differ at only two amino acids and baboon and rhesus P450c17 only at a single residue; the human/chimp enzyme differed from the baboon/rhesus enzyme by 25-27 residues (95% identity). Surprisingly, the greatest difference in enzymatic activities was a marked increase in 17alpha-hydroxylase activity of P450c17 in the baboon, which differs from rhesus only at residue 255 [arginine (Arg) in baboon, histine (His) in rhesus]. Residue 255 is also Arg in human and chimp. Wild-type human P450c17 and its Arg255His mutant had similar 17alpha-hydroxylase activities, but the Arg255Ala mutant had decreased 17alpha-hydroxylase activity. These data establish that Arg255 is important for 17alpha-hydroxylase activity and show that the evolution of adrenarche in higher primates is not determined by variations in the sequence of P450c17.