AbstractIn contrast with other animal species, humans possess three distinct genes for class I alcohol dehydrogenase and show polymorphic variation in the ADH1B and ADH1C genes. The three class I alcohol dehydrogenase isoenzymes share ∼93% sequence identity but differ in their substrate specificity and their developmental expression. We report here the first three‐dimensional structures for the ADH1A and ADH1C*2 gene products at 2.5 and 2.0 Å, respectively, and the structure of the ADH1B*1 gene product in a binary complex with cofactor at 2.2 Å. Not surprisingly, the overall structure of each isoenzyme is highly similar to the others. However, the substitution of Gly for Arg at position 47 in the ADH1A isoenzyme promotes a greater extent of domain closure in the ADH1A isoenzyme, whereas substitution at position 271 may account for the lower turnover rate for the ADH1C*2 isoenzyme relative to its polymorphic variant, ADH1C*1. The substrate‐binding pockets of each isoenzyme possess a unique topology that dictates each isoenzyme's distinct but overlapping substrate preferences. ADH1*B1 has the most restrictive substrate‐binding site near the catalytic zinc atom, whereas both ADH1A and ADH1C*2 possess amino acid substitutions that correlate with their better efficiency for the oxidation of secondary alcohols. These structures describe the nature of their individual substrate‐binding pockets and will improve our understanding of how the metabolism of beverage ethanol affects the normal metabolic processes performed by these isoenzymes.