The conversion of phenylalanine to tyrosine in mammalian tissues is catalyzed by a complex enzyme system composed of several essential enzymes and cofactors. All of these components have been assayed in liver biopsy samples from patients with the classic form of PKU. They are all present except for phenylalanine hydroxylase, thus establishing this enzyme as the missing component. This conclusion has been confirmed in immunotitration experiments with a specific antiserum to phenylalanine hydroxylase. With the use of a highly sensitive assay for the hydroxylase, 0.27% of the normal activity of phenylalanine hydroxylase has been detected in a liver sample from a patient with classic PKU. There is some evidence that this low level of catalytic activity is due to the presence of a nutant form of the enzyme rather than to very low levels of the normal enzyme. These results rule out the possibility that clasic PKU is caused by a deletion mutation. The finding that the properties of the enzyme are different from the normal enzyme also suggests that the low hydroxylase activity in PKU is not caused by a regulatory hene mutation, but rather by a mutation in the gene that codes for the structure of the hydroxylase.