Specific antibodies against l- erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l- erythro-biopterin distinguished among l- erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d- erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l- erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l- erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-( l- erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with 125I as the labeled ligand for the radioimmunoassay. BP- 125I-TYRA had similar binding affinity as the natural l- erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP- 125I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H + column was required for serum, cerebrospinal fluid, and brain tissues.