The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis‐acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T‐rich segment adjacent to the consensus elements that are present in the 5′ flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor.The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.