The range of phenylalanine hydroxylase activity was determined by measuring the conversion of radioactive phenylalanine to tyrosine in liver and kidney of various vertebrates. Rodents (rats, mouse, gerbil, hamster and guinea pig) were found to have the highest liver phenylalanine hydroxylase activity among all animals studied. They are also the only species that possessed a significant kidney phenylalanine hydroxylase activity which was about 25% of that found in the liver of the same animal. The synthetic dimethyl-tetrahydro-pteridine, used as a cofactor for the enzyme assay in most studies, catalyzed non-enzymatic hydroxylation of phenylalanine to tyrosine. Inclusion of boiled-blank and strict control of timing between incubation and product measurement were essential precautions to minimize erroneous results from substrate contamination and non-enzymatic hydroxylation.