Cyclic guanosine 3'-5'-monophosphate (cGMP) has recently been shown to constitute a second messenger for Xenopus laevis melatonin Mel1c receptors. To verify whether cGMP levels are also modulated by mammalian melatonin receptors, we cloned the genes encoding the human Mel1a and Mel1b receptor subtypes and expressed them in human embryonic kidney cells. Pharmacological profiles and inhibition of forskolin-stimulated adenosine 3'-5'-cyclic monophosphate levels by melatonin confirmed functional expression of high-affinity melatonin receptors. Mel1b receptor-transfected cells modulated cGMP levels in a dose-dependent manner via the soluble guanylyl cyclase pathway. In contrast, Mel1a receptors had no effect on cGMP levels. These results demonstrate that mammalian melatonin receptors modulate cGMP levels and reveal for the first time differences in signaling between melatonin receptor subtypes, which may explain the necessity to express different receptor subtypes.