Acetaldehyde, the first metabolite of ethanol, is implicated in several of ethanol’s actions, including the reinforcing and aversive effects. The neuronal mechanisms underlying acetaldehyde’s aversive effect, however, are poorly understood. The lateral habenula (LHb), a regulator of midbrain monoaminergic centers, is activated by negative valence events. Although the LHb has been linked to the aversive responses of several abused drugs, including ethanol, little is known about acetaldehyde. We, therefore, assessed acetaldehyde’s action on LHb neurons in rats. The results showed that intraperitoneal injection of acetaldehyde increased cFos protein expression within the LHb and that intra-LHb infusion of acetaldehyde induced conditioned place aversion in male rats. Furthermore, electrophysiological recording in brain slices of male and female rats showed that bath application of acetaldehyde facilitated spontaneous firing and glutamatergic transmission. This effect of acetaldehyde was potentiated by an aldeh...