Melatonin binding sites were characterized in human blood lymphocytes. The specific binding 2-[125I]iodo-melatonin ([125I]MEL) to human lymphocytes was dependent on time and temperature, stability, saturation, and reversibility. Moreover, guanine nucleotides decreased the specific binding of [125I]MEL to crude membranes of human lymphocytes, suggesting the coupling of these binding sites to a guanosine nucleotide binding regulatory protein(s). In competition studies, the specific binding of [125I]MEL to lymphocytes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that data were compatible with the existence of two classes of binding sites: a high-affinity site with a Kd of 5.20 +/- 0.79 nM and a binding capacity of 50.6 +/- 11.0 fmol/10(7) cells, and a low-affinity site with a Kd of 208.5 +/- 50.2 nM and a binding capacity of 2691 +/- 265 fmol/10(7) cells. However, concentration-dependent binding of [125I]MEL to lymphocytes was saturable and resulted in a linear Scatchard plot, suggesting binding to a single class of binding sites. The Kd for the single site was 1.02 +/- 0.34 nM with a binding capacity of 10.1 +/- 1.6 fmol/10(7) cells. Their affinities closely correlated with the production of cyclic nucleotides, suggesting a physiological role for the melatonin binding sites. Thus, melatonin potentiated the effect of vasoactive intestinal peptide (VIP) on cyclic AMP production (ED50 = 1.9 nM) and stimulated cyclic GMP accumulation (ED50 = 125 nM). Results demonstrate the existence of two binding sites for [125I]MEL in human blood lymphocytes, with a high-affinity binding site coupled to the potentiation of the effect of VIP on cyclic AMP production and a low-affinity binding site coupled to activation of cyclic GMP production.