Melatonin regulates circadian and seasonal physiology via melatonin receptors expressed in the brain. However, little is known about the signal transduction mechanisms that mediate the action of melatonin in neuronal cells. To begin to address this issue, we expressed the human MT(1) receptor in the human neuroblastoma SH-SY5Y cell line. In this cell line, melatonin acutely stimulated cAMP synthesis through a calcium-calmodulin dependent pathway. This stimulatory effect was independent of an interaction with G(i) or G(s) G proteins and dependent upon internal calcium stores. Melatonin also potentiated forskolin-activated cAMP synthesis. Differentiation of the neuroblastoma cells with retinoic acid to the neuronal phenotype did not alter the ability of melatonin to acutely stimulate cAMP. These data may be relevant to the neuronal action of melatonin and highlight the importance of the cellular context of expression of melatonin and other G protein-coupled receptors.