Isozyme phenotypes of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from human gastroendoscopic as well as surgical gastric biopsies were determined by starch gel electrophoresis and agarose isoelectric focusing. gamma gamma ADH isozymes were expressed predominantly in the mucosal layer of the stomach, whereas beta beta isozymes were in the muscular layer. In the 56 gastroendoscopic mucosal biopsies examined, the homozygous ADH3 1-1 phenotype was found in 75% of the samples, and the heterozygous ADH3 2-1 phenotype in 25%. Accordingly, the gene frequencies of the alleles ADH1/3 and ADH2/3 were calculated to be 0.88 and 0.12, respectively. Using a modified agarose isoelectric focusing procedure, gastric ALDH I, ALDH II, and up to five ALDH III forms could be clearly resolved. The ALDH III isozymes accounted for more than 80% of the total ALDH activities in gastric mucosa and exhibited Km values in the millimolar range for propionaldehyde at pH 9.0. Forty-five percent of the 55 gastroendoscopic biopsies studied lacked ALDH I isozyme. The complex gastric ALDH III isozyme phenotypes seen in these biopsies fall into three patterns. They can be interpreted by a genetic hypothesis, based on a dimeric molecule, in which there are two separate genes, ALDH3a and ALDH3b, with the ALDH3b locus exhibiting polymorphism. The homozygous phenotypes ALDH3b 1-1 and ALDH3b 2-2 were found to be 4 and 76%, respectively, and the heterozygous ALDH3b 2-1 phenotype 20%, of the total. Therefore, the allele frequencies for ALDH1/3b and ALDH2/3b were calculated to be 0.14 and 0.86, respectively. Several lines of biochemical evidence consistent with this genetic model are discussed.