The aims of this study were to characterize the melatonin receptors in rat brain arteries forming the circle of Willis. Saturation studies performed using in vitro autoradiography and [125I]iodomelatonin revealed the presence of two binding sites: one with a Kd of 13 pM, and the second characterized by a Kd of 832 pM. Coincubation with a nonhydrolyzable guanine nucleotide analog [guanosine-5'-O-(3-thiotriphosphate)] inhibited 2-[125I]iodomelatonin binding in a concentration-dependent manner, whereas adenine nucleotide adenosine-5'-O-(3-thiotriphosphate) was ineffective. In saturation studies performed in the presence of guanosine-5'-O-(3-thiotriphosphate), the high affinity site was no longer detectable, and the affinity of the receptor was decreased to the high picomolar range. Melatonin, at nanomolar concentrations, was able to inhibit forskolin-stimulated cAMP production in rat circle of Willis arteries. Preincubation with pertussis toxin counteracted the effect of melatonin. Our results demonstrate that melatonin receptors in rat cerebral arteries are linked to their second messenger through a pertussis toxin-sensitive G-protein, similar to what has been described for melatonin receptors in different areas of vertebrate brain.