Melatonin is a potent free radical scavenger, antioxidant and neuroprotective drug. On the other hand, galantamine is a cholinergic drug with antioxidant and neuroprotective properties linked to inhibition of acetylcholinesterase and allosteric modulation of nicotinic receptors. This investigation evaluated a possible synergistic neuroprotective effect of subeffective concentrations of combined galantamine and melatonin. Human neuroblastoma SH-SY5Y cells were subjected to a mitochondrial oxidative stress, by blockade of mitochondrial complexes I and V with rotenone and oligomycin-A (R/O); cells were treated for 24 hr with R/O. This caused 40% of the cell to die as measured by lactate dehydrogenase (LDH) release. Cell incubation with increasing concentrations of galantamine (10-300 nm) or melatonin (0.3-10 nm) for 24 hr, followed by a 24-hr period with R/O, caused a concentration-dependent protection; maximum protection was achieved with 300 nm galantamine (56% protection) and 10 nm melatonin (50% protection). Combination of subeffective concentrations of melatonin (0.3 nm) and galantamine (30 nm) caused a synergistic and significant protection that was similar to the maximum protection afforded by effective concentrations of melatonin or galantamine alone. This protective effect was completely reversed when nicotinic and melatonin receptors were blocked respectively by mecamylamine and luzindole. The neuroprotective effect was prevented by chelerythrine, LY294002, and Sn (IV) protoporphyrin IX dichloride (SnPP), indicating the participation of the PKC/PI3K/Akt activation and induction of the antioxidant enzyme heme oxygenase-1. The synthesis of novel multitarget compounds having in a single molecule the combined neuroprotective properties of galantamine and melatonin could be a new strategy for potential therapeutic agents in neurodegenerative diseases.