Ethanol (EtOH) alters neural activity through interaction with various neurotransmitters and neuromodulators. The endogenous opioid system seems to play a key role in the activities of EtOH, since the opioid antagonist naltrexone (ReVia^®) attenuates craving. We have investigated the transcriptional regulation of opioid system genes in response to EtOH exposure for up to 96 h in human neuroblastoma SH-SY5Y cells using quantitative real-time polymerase chain reaction. We observed a significant decrease in the expression of opioid peptide precursors (proopiomelanocortin, proenkephalin, and prodynorphin) and of the kappa opioid receptor after 48 and 72 h of EtOH exposure (10 and 40 mM). These alterations were not present when the EtOH metabolism was blocked by 4-methylpyrazole. To evaluate whether the effects evoked by EtOH were possibly due to the first product of EtOH metabolism, cells were exposed to 0.4 mM acetaldehyde. We observed the same pattern of changes for prodynorphin, proenkephalin, and the kappa opioid receptor as after 72 h exposure to EtOH. These results contribute to our understanding of EtOH action at a cellular level and provide evidence of the role of acetaldehyde in mediating some of the EtOH-induced effects.—D`Addario, C., Ming, Y., Ögren, S-O., Terenius, L. The role of acetaldehyde in mediating effects of alcohol on expression of endogenous opioid system genes in a neuroblastoma cell line.