N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was prepared and used to identify melatonin binding proteins in rat brain synaptosomes. Incubation of the synaptosomes with BIM resulted in a time and concentration dependent, irreversible inhibition of 2-[125I]iodomelatonin binding. In parallel, the radioactive form of BIM, N-bromoacetyl-2-[125I]iodo-5-methoxytryptamine ([125I]BIM) became incorporated into the synaptosomes. The incorporation of [125I]BIM was inhibited by BIM, 2-iodomelatonin and melatonin but not by 5-methoxytryptamine or N-acetyl serotonin. [125I]BIM became covalently attached to three polypeptides with apparent molecular weight values of 92, 55 and 45 kDa; the labeling of all three proteins was markedly inhibited by melatonin. These results indicate that the 92, 55 and 45 kDa polypeptides are melatonin binding proteins.