P450c17 is the single enzyme that catalyzes steroid 17-hydroxylase and 17,20 lyase activities, and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. While both activities are catalyzed on a single active site, the ratio of these activities is regulated by post-translational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8Br-cAMP. Key components of the ERK 1/2 and MEK 1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the PKA/PI3K/Akt pathway and the calcium/calmodulin/MEK pathway had no effect on the ratio of 17, 20 lyase activity to 17-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the ROCK/rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a ‘priming phosphorylation’.