Class 3 aldehyde dehydrogenase (ALDH-3) is induced by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and during chemical carcinogenesis. These inductions as well as the basal expression of ALDH-3 vary significantly in different organs. In order to identify DNA elements controlling ALDH-3 expression, we have cloned and analyzed approximately 5.5 kb of the 5' flanking region of the ALDH-3 gene. Deletion analysis showed that the 5' flanking region contains at least three functional domains: a strong promoter proximal to the transcription start site, inhibitory regions upstream of the promoter, and TCDD-responsive enhancers. The TCDD-responsive enhancers in the ALDH-3 gene were functionally similar to xenobiotic responsive elements in the cytochrome P450IA1 gene. These results indicate that transcription of the ALDH-3 gene is controlled by cooperation of at least three functional domains.