Previous studies revealed polymorphisms in mitochondrial aldehyde dehydrogenase (ALDH2) and the major histocompatibility complex RT1.A that appeared in association with alcohol drinking preference in the alcohol-preferring (P) and alcohol-nonpreferring (NP) rat lines. To determine the strength of these associations, the P and NP lines were crossed, and cosegregation studies were performed in the F2 progeny. The ALDH2Q allele and the ALDH2R allele, found in the P and NP lines, respectively, were found not to be associated with the high and low drinking animals in the F2 progeny. Kinetic studies with ALDH2Q and ALDH2R isozymes purified from the mitochondria of P and NP rat livers, respectively, showed that the polymorphism would not be expected to be associated with altered aldehyde metabolism. The association between RT1.A and alcohol preference (RT1.Auu with high and RT1.AII with low alcohol intake) also could not be confirmed in the segregating F2 progeny of the P x NP intercross.