The class I hepatic alcohol dehydrogenases (ADHs) are primarily responsible for ethanol metabolism in humans. Genetic polymorphism at the ADH2 locus results in the inheritance of isozymes of strikingly different catalytic properties. The most common ADH2 allele, ADH2*1, encodes the low Km isozyme subunit β1. The ADH2*3 allele encodes a high-activity isozyme subunit of alcohol dehydrogenase, β3, identified in ∼25% of African-Americans. The Vmax of β3β3-ADH is 30 times greater than that of the β1β1-ADH. Therefore, we hypothesized that the rate of ethanol metabolism, an important factor in the toxicity of ethanol, in persons with β3-containing ADH, either β3β3- or β1β3-ADH, would be faster than that of persons with only β1β1-ADH. We tested this hypothesis with ethanol administered orally to healthy, young African-Americans. Three hundred and twenty-six African-American men and women were genotyped using polymerase chain reaction amplification of their leukocyte DNA followed by hybridization with allele-specific probes. One hundred twelve volunteers, selected by genotype, received an oral dose of ethanol designed to produce a blood ethanol concentration of 80 mg/dl (0.080 g/dl), when the blood alcohol concentration-time curve was extrapolated back to time 0. Ethanol metabolic rates (β60s) were determined in the 112 subjects from the slope of the pseudolinear portion of the blood ethanol concentration-time curves. The mean β60 of African-Americans having β3-containing ADH isozymes had significantly faster ethanol elimination rates than those with only β1β1-ADH isozymes. There were no significant differences in body weight, ethanol intake in the week before testing, peak breath ethanol concentration, time to peak, or volume of distribution between the genotype groups. Within each of these groups, men had lower ethanol disappearance rates than women. These results demonstrate in vivo the kinetic differences of ADH2 isozymes that may influence individual risk for the effects of ethanol.