The 5'-flanking regions of the gene encoding mitochondrial adenylate kinase isozyme 3(AK3) of the human and the bovine have been characterized. We have isolated the 5'-flanking region of the human AK3 gene and compared it with that of the bovine AK3 gene isolated previously. Sequence comparison of promoter regions of the human and bovine genes revealed four homologous DNA sequences in addition to common structural characteristics that both promoters lack typical TATA and CAAT boxes and have high G+C contents. We measured the promoter activities in HeLa cells of the constructs containing the deleted promoter regions and the chloramphenicol acetyltransferase(CAT) gene. From the results obtained the minimum promoter region was characterized. In the presence of short DNA segments of the bovine AK3 promoter region, the transcriptional activity in HeLa cells of both the human and bovine genes varied depending on the segments used. The results suggested that the segments containing the homologous sequences mentioned above act as positive regulatory elements and that proteins in HeLa cells commonly participate in transcriptional regulation of both the AK3 genes.