Phenylketonuria (PKU) was induced in rats by the combined feeding of 3 per cent excess phenylalanine and 0.12 per cent of p-chlorophenylalanine, an inhibitor of phenylalanine and tryptophan hydroxylases. Increased concentrations of phenylalanine and increased ratio of phenylalanine to tyrosine were demonstrated in blood from pregnant rats fed the experimental PKU diet from day 10 to 20 of pregnancy, in fetal blood and amniotic fluid of fetal animals from mothers fed the PKU diet, and in blood of rats fed the PKU diet for 28-30 days beginning at 20-21 days of age. Both phenylpyruvic acid and orthohydroxyphenylacetic acid were excreted by rats fed the PKU diet, but neither were detected in urine in animals fed either excess phenylalanine or excess inhibitor alone. Reduced serotonin concentrations were found in brains of rats fed p-chlorophenylalanine, either alone or in combination with excess phenylalanine in the PKU diet. These biochemical changes in rats with induced PKU and the behavioral changes described earlier are similar to those of the human condition. The animal model should prove useful in searching for the mechanism of the disease.