A reverse-phese high-performance liquid chromatography (hplc) procedure was developed for the separation of biopterin from other unconjugated pteridines. Using iodine in 0.1 n HCl, 7,8-dihydrobiopterin, and tetrahydrobiopterin were shown to be effectively oxidized to biopterin. When 7,8-dihydrobiopterin was oxidized with iodine in 0.1 n NaOH, 90% was recovered as biopterin. Using these same conditions, the oxidation of tetrahydrobiopterin resulted in an 80% conversion of tetrahydrobiopterin to pterin (2-amino-4-hydroxy-pteridine). Based upon these chemical properties of the reduced forms of biopterin, a microanalytical procedure was developed for the quantitative analysis of the reduced forms of biopterin in biological materials. This procedure consists of an initial iodine oxidation of the reduced forms of biopterin, a partial purification on a Dowex 50 column and subsequent hplc analysis. Using fluorescence detection in the hplc analysis, the sensitivity of this procedure permits the quantitation of biopterin at picomole levels. Using our procedure in the analysis of biological samples, biopterin in rat tissues (pineal, liver, adrenal, brain, and blood) was shown to be present predominantly in the tetrahydro form. Both biopterin and neopterin were present in human blood and urine. The synthesis and secretion of biopterin by the kidney is discussed, as well as a possible role for tetrahydroneopterin as a cofactor in the enzymatic hydroxylation of aromatic amino acids in man.