The two major forms of rat liver phenylalanine hydroxylase have been isolated and partially purified. The tetrahydrobiopterin-dependent activity of these forms can be differentially stimulated by exposure to enzymatic phosphorylating conditions. This in vitro treatment is associated with incorporation of 32p into the enzymes and generates a further, chromatographically distinct, species. These results suggest that the multiple forms of rat liver phenylalanine hydroxylase are due to different degrees of phosphorylation.