The direct action of pineal melatonin on the renal system is supported by our demonstration of 2-[125I]iodomelatonin binding sites in the male guinea pig kidney. Scatchard analyses and Hill coefficients revealed a single type of binding site with an equilibrium dissociation constant (Kd) of 22.3 +/- 1.6 pmol/l and a maximum binding density (Bmax) of 0.99 +/- 0.03 fmol/mg protein (n = 7) at mid-light. There was no significant difference in the Kd and Bmax values between kidney tissues collected at the middle of light and dark periods. The pharmacological profile of these 2-[125I]iodomelatonin binding sites indicated high specificity for melatonin, 2-iodomelatonin and 6-chloromelatonin while kinetic studies generated a Kd value of 28.4 +/- 7.3 pmol/l (n = 5) which was comparable to that determined from Scatchard transformations. Our results suggest that these binding sites are stable, reversible, saturable, specific, and of high affinity. Regional distribution study showed that specific binding of 2-[125I]iodomelatonin was 8-fold higher in the cortical region than that in the medullary region. Studies of subcellular distribution showed that 59.3% of binding sites were localized in crude nuclear fractions followed by crude mitochondrial fractions (22.3%) and crude microsomal fractions (18.3%) with no detectable binding in cytosolic fractions. Our present findings suggest the presence of putative melatonin receptors in the guinea pig kidney, which support the hypotheses of melatonin-regulated renin secretion together with renal excretory functions via melatonin receptors.