Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca 2 + /calmodulin-dependent protein kinase II and protein kinase C (Ca 2 + /phospholipid-dependent protein kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 +/- 0.05 (n = 4) and 0.74 +/- 0.03 (n = 5) 3 2 P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent protein kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca 2 + /calmodulin-dependent protein kinase II. Phosphorylation of sepiapterin reductase by these protein kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca 2 + -activated protein kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.