The lyase activity of human CYP17 (17alpha-hydroxylase-17,20-lyase also P-450c17 or P-45017alpha) is greatly dependent on the presence of cytochrome b5, and this effect has been ascribed an important regulatory role [Lee-Robichaud, Wright, Akhtar and Akhtar (1995) Biochem. J. 308, 901-908]. This facet was further investigated by site-directed mutagenesis of selected basic residues of human CYP17. The purified mutant proteins were subjected to detailed kinetic analysis. It was found that the mutation of Lys83, Arg347 and Arg358 produced proteins that were deficient in their responsiveness to cytochrome b5, and the effect was most pronounced for the two arginine mutants (Arg347-->His and Arg358-->Gln) which have been found in male patients suffering from genital ambiguity. These residues are invoked to mediate protein-protein interaction between cytochrome b5 and CYP17, which 'awakens' the lyase activity of the enzyme required for androgen formation.