The pattern of unconjugated pterins in liver tissue and in urine from patients with atypical forms of phenylketonuria with hyperphenylalaninemia (HPA) has been investigated with a high performance liquid chromatographic technique. Two patients with defects in the biosynthesis of biopterin have been shown to have higher than normal levels of neopterin and lower than normal levels of biopterin. In contrast, a patient with HPA due to a deficiency of dihydropteridine reductase has the reverse urinary pattern, i.e., high biopterin, low neopterin. These results indicate that the ratio of neopterin to biopterin in urine can be of value in discriminating between HPA due to a deficiency of phenylalanine hydroxylase (classic PKU), HPA due to dihydropteridine reductase deficiency, and HPA due to a block in the biosynthesis of biopterin.