The gastrointestinal tract is a major site of alcohol dehydrogenase (ADH) activity in humans and rodents. Because class I ADH (ADH-I) and class IV ADH (ADH-IV), but not class III ADH (ADH-III), function as retinol dehydrogenases in vitro and may thus participate in retinoid signaling needed for epithelial differentiation, the aim of this study was to determine the localization of these enzymes along the gastrointestinal tract. Specific antibodies were used to examine the tissue distribution of all three known classes of mouse ADH by Western blotting, and cellular localization was determined by immunohistochemistry. ADH-I was detected primarily in the intestine, liver, kidney, adrenal, and uterus, with detection of ADH-III in all tissues examined, and detection of ADH-IV primarily in the esophagus, stomach, adrenal, skin, ovary, and epididymis. Along the gastrointestinal tract, ADH-III was not specifically localized, whereas ADH-I was localized exclusively in the villus epithelium of the small intestine and absorptive epithelium of the large intestine, with ADH-IV being localized exclusively in the basal and suprabasal epithelial cells of the esophagus and gastric pit surface epithelium of the stomach. The ADH localization patterns observed are consistent with ADH-I and ADH-IV, but not ADH-III, functioning physiologically in retinol metabolism needed for epithelial differentiation. Our results further suggest that the functions of ADH-I and ADH-IV are regionally restricted to the lower and upper components, respectively, of the gastrointestinal epithelium, a finding that may relate to the different efficiencies of these two enzymes for retinol oxidation, as well as to the different susceptibilities of the upper and lower digestive tracts for ethanol-induced cancers.