The effect of phenylalanine on melanogenesis was studied in the RPMI-3460 hamster melanoma cell line. L-phenylalanine was a relatively weak competitive inhibitor of the tyrosinase activity in pigment cell homogenates but D-phenylalanine did not affect this tyrosinase activity. In vivo, 10 mM L- or D-phenylalanine inhibited the incorporation of labeled tyrosine into melanin. Since phenylalanine reduced the size of the acid-soluble intracellular tyrosine pool relative to controls, this reduction is proposed as the cause of the diminished incorporation of tyrosine into melanin. In addition, melanin synthesis generated from endogenous tyrosine, in the absence of tyrosine in the culture medium, was not affected by the presence of L- or D-phenylalanine. Phenylalanine-related depression of melanin synthesis in cell culture therefore, was not caused by inhibition of tyrosinase activity, but by restriction of tyrosine uptake by phenylalanine. The results with this mammalian cell model suggest that the pathologic phenylalanine:tyrosine imbalance found in phenylketonuria (PKU) may cause a reduction of melanin synthesis in hair follicles and normally pigmented areas of the brain by affecting uptake of tyrosine by melanocytes. The reduced pigmentation in PKU is more likely to be caused by reduced serum tyrosine levels and by competition for tyrosine uptake with phenylalanine than by phenylalanine inhibition of tyrosinase activity.