By using 2-[125I]iodomelatonin receptor binding studies, we have previously demonstrated high affinity melatonin receptors, the binding activities of which are regulated by testosterone, in the corpus epididymis of rats. In this report, some of the basic molecular and cellular characteristics of these high affinity melatonin receptors in rat corpus epididymis were analyzed. MEL1A and MEL1B receptor mRNAs were expressed by rat corpus epididymal epithelial cells as revealed by in situ hybridization. Functionally, these high affinity melatonin receptors are negatively coupled to adenylyl cyclase via pertussis toxin (PTX) sensitive Gi protein and the inhibitory effects of melatonin on forskolin-stimulated cAMP accumulation were enhanced by 5alpha-dihydrotestosterone (5alpha-DHT). Interestingly, opposing interactions between melatonin and beta-adrenergic receptor signaling in rat epididymal epithelial cells were observed with melatonin inhibiting norepinephrine- and isoproterenol-stimulated cAMP accumulation. In conclusion, our data support a modulatory action of melatonin, mediated via pertussis toxin-sensitive Gi-coupled MEL1A and MEL1B receptors, in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions.