The effect of guanine nucleotides on the binding of 2-[125I]iodomelatonin in membrane preparations of guinea pig spleen was studied. The GTP analogues guanosine 5’-0-(3-thiophosphate) and 5’-guanylimidodiphosphate dose-dependently inhibited the binding. Saturation studies revealed that the presence of GTP analogues either increased the equilibrium dissociation constant (Kd) alone or both increased the Kd and decreased the binding site density (Bmax). Our results suggest that melatonin receptors in the guinea pig spleen, similar to those in the neural and retinal tissues, are coupled to a G protein. This study showed that Na+ and Li+ dose-dependently inhibited 2-[125I]iodomelatonin binding while Mg2+ potentiated the binding. K+ and choline were without significant effects. Low Ca2+ concentrations (1–5 mmol/l) potentiated the radioli-gand binding while higher concentrations were inhibitory. Saturation studies demonstrated that 125 mmol/l Na+ decreased the Bmax while 2.4 mmol/l Ca2+ increased the Bmax and 40 mmol/l Ca2+ increased the Kd. Our results suggest that physiological concentrations of Na+ and Ca2+ may play an important modulatory role on melatonin binding to its receptors in the s