Purified cholesterol 7α-hydroxylases (C7αH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7αH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7αH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7αH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7αH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7αH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7αH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7αH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7αH proteins by cAMP-dependent protein kinase increased C7αH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7αH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7αH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.