The anti-apoptotic effect of melatonin has been described in vivo and in vitro. A previous report has revealed that melatonin suppresses nitric oxide (NO)-induced apoptosis via the induction of Bcl-2 expression in PGT-beta pineal cells. To investigate the protective mechanism of melatonin on NO donor S-nitroso-N-acetyl-penicillamine (SNAP)-induced apoptosis, we examined the anti-apoptotic upstream signaling pathway of Bcl-2 in the human neuroblastoma cell line SK-N-MC. The flow cytometry results revealed that apoptosis occurred in NO-treated cells, while cell death was inhibited by pretreatment with melatonin (100 microm). In addition, decreased Bax expression, increased Bcl-2 expression and a decreased release of cytochrome c into the cytosol were observed in the melatonin-pretreated SK-N-MC cells. We also found that melatonin treatment induced the activation of Akt/PKB and the phosphorylation of GSK3alpha/beta and Bad. Furthermore, melatonin treatment not only increased the protein-protein interactions between 14-3-3beta and p-Bad, but also decreased the release of cytochrome c from mitochondria into the cytosol. In summary, the protective effect of melatonin against NO-induced apoptosis was mediated by the inhibition of Bad translocation from the cytosol to the mitochondria by the induction of protein-protein interactions between 14-3-3beta and p-Bad.