Specific antibodies against Derythroneopterin have been pmpared in rabbits using a conjugate of Derythroneopterin to bovine serum albumin (~rythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with lzsl as the labeled ligand NP-[izSI]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with Buorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak. Approximately 10 pmol/g tissue of neopterin was detected by this radioimmunoassay in rat tissues (liver, kidney, adrenal gland, and brain). About 35% less neopterin value was obtained when the samples from human urine were oxidized in alkaline solution, suggesting the presence of alkaline-labile reduced forms of neopterin. When the extract of rat liver was treated with alkaline phosphatase prior to the acid oxidation process, total neopterin values were increased approximately twofold. This indicates that approximately 50% of neopterin in rat liver is present as a phosphate ester.