Human phenylalanine hydroxylase is in an activated state compared to its rat counterpart. Our finding suggests that the N-terminal domain is responsible for maintaining the human enzyme in an activated state. Phosphorylation of a serine residue at the regulatory domain of the rat liver phenylalanine hydroxylase results in activation of the enzyme. Substitution of the serine residue with negatively-charged amino acid residues results in activation of the enzyme to the same extent as does the phosphorylation of this residue by cAMP-dependent protein kinase A. This study has unequivocally demonstrated that activation by phosphorylation of the serine residue of the rat liver hydroxylase is due to the introduction of negative charge(s).