The two major forms of rat liver phenylalanine hydroxylase have been resolved by calcium phosphate chromatography and have been highly purified. These different forms of the enzyme contain dissimilar amounts of endogenous protein-bound phosphate. The hydroxylase activity of these forms, as assayed in the presence of tetrahydrobiopterin, can be differentially stimulated by treatment with Mg2+, ATP, cyclic AMP, and protein kinase, leading to the production of a chromatographically distinct species. In the presence of [gamma-32P]ATP, activation is accompanied by differential incorporation of 32P into the subunits (Mr = 50,000) of the two enzyme forms leading to a new form which contains 1 mol of phosphate/subunit. In addition, phosphorylation in vitro eliminates the electrophoretic heterogeneity displayed by the isolated forms. These data show that the multiple forms and electrophoretic heterogeneity of phenylalanine hydroxylase are correlated with the degree of phosphorylation and also provide further evidence for the regulation of this enzyme by phosphorylation and dephosphorylation.