We previously reported that an NAD-dependent in situ retinoic acid supply system, which comprises some isoforms of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) and provides retinoic acid from retinol via a 2-step oxidation process, exists in the rat esophagus. Herein, their isoforms responsible for the pathway and its localization in the rat esophagus was examined. The expressions of mRNAs of various isoforms of ADH and ALDH were examined in the fraction mainly comprising mucosal layer of the rat esophagus by RT-PCR. Expression levels of Class IV ADH and ALDH 1A1 were compared between the fractions and that mainly comprising muscle layer of the rat esophagus by quantitative PCR. The catalytic activities producing retinoic acid from retinal were compared between the 2 fractions and its optimum pH was also determined. Classes I, III, and IV ADHs and ALDHs 1A1 and 3A1 were predominant isoforms in the rat esophageal mucosa. The expression levels of mRNA of Class IV ADH and ALDH 3A1 were significantly higher in the mucosal than in the muscle layer. Consistently, the catalytic activities producing retinoic acid from retinal were significantly higher in the former than the latter. The optimum pH of the process was 9.0. Considering the affinities for retinol and retinal of ADHs and ALDHs expressed in the rat esophagus, the NAD-dependent in situ retinoic acid supply system in the rat esophagus is thought to comprise Class IV ADH and ALDH 1A1. In the rat esophagus, the system exists predominantly in the mucosal layer.