The development pattern of phenylalanine hydroxylase was studied in rat kidney. Traces of activity were observed on the 20th and 21st days of gestation, but significant amounts appeared first on the 22nd day. Kidney actvity showed an optimum at pH 7.0, similar to liver. Separation of isozymes of phenylalanine hydroxylase from rat kidney will permit us to determine if the properties of the kidney enzyme are similar to those of the liver enzyme. The assay for phenylalanine hydroxylase was modified to minimize interference from non-enzymatic conversion of phenylalanine to tyrosine. Phenylalanine hydroxylase activity was measured in a variety of species including salamander, fish, frog, pig, cow, as well as rodents, primates, and man. Rodents are unique in having high activity in liver and in the presence of enzyme in kidney. Assay of activity in human fibroblast cultures will be undertaken using tetrahydrobiopterin. Affinity chromatography using a pteridine column will be used to isolate phenylalanine hydroxylase.