Determination of melatonin by radioimmunoassay in plasma samples from hemolyzed blood often yields flawed values. We studied the possibility that hemoglobin can bind melatonin and the iodinated tracer 125I-melatonin. The specific binding of 125I-melatonin to purified bovine hemoglobin was found to be rapid, saturable, and reversible (K(d) = 315 pM, B(max) = 58 pmol/mg protein) and was inhibited by 2-iodomelatonin, serotonin, melatonin, and 5- methoxytryptamine. These data are compatible with the concept that hemoglobin can interfere with melatonin determinations by competing for melatonin and the iodinated tracer. Unlike melatonin receptor binding, the binding of 125I-melatonin to hemoglobin was not inhibited by guanine nucleotide analogs (i.e., GTPγS, GTPβS, and Gpp(NH)p). Sodium cyanide had no effect on 125I-melatonin binding, indicating that 125I-melatonin does not bind to the heme group. On the other hand, 2, 3-bisphosphoglycerate, at physiological concentrations (3-4 mM), decreased the apparent B(max) and K(d) of 125I- melatonin binding to hemoglobin. These data suggest that 125I-melatonin binding to hemoglobin is conformation specific and is unfavorable in the deoxyhemoglobin state. Hemoglobin may serve as a carrier protein for melatonin in the blood and discharge it in the target organs. Subsequently, the efficacy of melatonin's action as a hormone or antioxidant in target tissues may be enhanced.