During our continuing study of pteridine metabolism, the need arose for a more rapid and quantitative determination of pterins in biological fluids. By adopting and modifying previously developed techniques, we have obtained a rapid and sensitive method that allows the simultaneous determination of eight different pterins in human urine and blood. When examined over a 10-day period, the levels of pterins excreted by a normal individual averaged the following values expressed in picomoles per mg of creatinine: biopterin, 9104; neopterin, 6018; xanthopterin, 6561; pterin, 1136; isoxanthopterin, 636; pterin-6-carboxylate, 483; and 6-hydroxymethylpterin, 315. Moreover, 6-hydroxymethylpterin and pterin-6-carboxaldehyde were detected for the first time in the blood of normal individuals.