Melatonin is present in human semen, and may affect sperm motility. The presence of melatonin receptors on spermatozoa has not yet been reported. Detection of melatonin-binding sites may be limited because of the masking of such sites by sialic acid. Spermatozoa were obtained from eligible human donors, incubated with neuraminidase to remove sialic acid residues, and saturation binding assays were carried out using 2- 125I-melatonin as a receptor probe. Consistent 125I-melatonin binding could only be obtained after spermatozoa were treated with neuraminidase. Scatchard analysis revealed a low-affinity binding site (ML-2) with a K d value of 127 ± 6 nM and a B max of 25 ± 4.5 fmol/mg protein. These results present evidence of melatonin-binding sites in spermatozoa. Sialic acid possibly regulates the binding of melatonin to these sites.