Dependence on alcohol, a most widely used drug, has a heritability of 50-60%. Wistar-derived rats selectively bred as low-alcohol consumers for many generations present an allele (Aldh2(2)) of mitochondrial aldehyde dehydrogenase that does not exist in high-alcohol consumers, which mostly carry the Aldh2(1) allele. The enzyme coded by Aldh2(2) has a four- to five-fold lower affinity for NAD than that coded by Aldh2(1). The present study was designed to determine whether these polymorphisms account for differences in voluntary ethanol intake and to investigate the biological mechanisms involved. Low-drinker F0 Aldh2(2)/Aldh2(2) rats were crossed with high-drinker F0 Aldh2(1)/Aldh2(1) rats to obtain an F1 generation, which was intercrossed to obtain an F2 generation that segregates the Aldh2 alleles from other genes that may have been coselected in the breeding for each phenotype. Data show that, with a mixed genetic background, F2 Aldh2(1)/Aldh2(1) rats voluntarily consume 65% more alcohol (P<0.01) than F2 Aldh2(2)/Aldh2(2) rats. A major phenotypic difference was a five-fold higher (P<0.0025) peak blood acetaldehyde level following ethanol administration in the lower drinker F2 Aldh2(2)/Aldh2(2) compared to the higher drinker F2 Aldh2(1)/Aldh2(1) animals, despite the existence of identical steady-state levels of blood acetaldehyde in animals of both genotypes. Polymorphisms in Aldh2 play an important role in: (i) determining peak blood acetaldehyde levels and (ii) modulating voluntary ethanol consumption. We postulate that the markedly higher peak of blood acetaldehyde generated in Aldh2(2)/Aldh2(2)(2) animals is aversive, leading to a reduced alcohol intake in Aldh2(2)/Aldh2(2) versus that in Aldh2(1)/Aldh2(1) animals.