The relationship between high dietary levels of aromatic amino acid and regulation of pteridines in Drosophila eyes was examined by measuring changes in pool levels of six pterins in the wild type and mutants and amino acid pool levels in flies that carry mutations for pteridine biosynthesis. The effect upon relative viability and developmental times was also analyzed; relative viability was affected by L-phenylalanine, L-tryptophan, and L-tyrosine in decreasing order and the D-amino acids had little or no effect. The changes in concentration of biopterin, dihydrobiopterin, pterin, sepiapterin, drosopterins, and isoxanthopterin showed a characteristic pattern of increased and/or decreased amounts in response to each of the three L-amino acids. Pterin was regularly increased, and isoxanthopterin decreased. L-Tyrosine caused a 2.1-fold increase in dihydrobiopterin, the largest increase found in this study; L-tryptophan also caused dihydrobiopterin to increase but L-phenylalanine did not. Of 18 eye-color mutants examined, 2 were found to contain high levels of phenylalanine and/or tyrosine, Pu2 and Hnr3. These two mutants, along with prc4 cn/prm2b cn, were shown to be very sensitive to dietary L-phenylalanine, indicating that having low levels of certain pteridines makes them susceptible to toxic effects of these amino acids. Therefore, high levels of aromatic amino acids can perturb the balance among pteridine pools, and low levels of some pteridines in mutants are correlated with the inability to withstand the toxic effects of phenylalanine. From the patterns of change in the pteridines we suggest that tetrahydropterin may also be a cofactor for hydroxylation of phenylalanine, along with tetrahydrobiopterin.