L-Phenylalanine was administered intraperitoneally, intravenously, intramuscularly and orally to rats, and its metabolites were separated and identified by high-performance liquid chromatography (HPLC) with fluorescence detection. Chromatographic peaks were identified on the basis of their retention behavior and the assignment of peaks of HPLC was verified by ion-exchange chromatographic analysis. The three metabolites p-, m- and o-tyrosines were formed in vivo after phenylalanine administration. The extents of formation of m- and o-tyrosines were significantly increased by the administration of L-phenylalanine. However, the concentrations of m- and o-tyrosines in the serum were reduced by the administration of α-methyltyrosine, which is an inhibitor of tyrosine hydroxylase. On the other hand, the inhibition of phenylalanine hydroxylase activity by the administration of p-chlorophenylalanine and ethionine resulted in a marked increase of the concentration of phenylalanine in the serum, and the formation of m- and o-tyrosines was also significantly increased. These results suggest that the formation of m- and o-tyrosines from phenylalanine is caused mainly by tyrosine hydroxylase, but not by phenylalanine hydroxylase.