The gene structure of the human ATP synthase alpha subunit (hATP1) was determined by cloning and sequencing. This gene is approximately 14 kbp in length and contains 12 exons interrupted by 11 introns. Mapping of the clones of hATP1 and Southern blot analysis of the genomic gene showed that there were a single copy of bona fide hATP1 gene and two pseudogenes. Primer extension and S1 mapping analysis showed the presence of multiple transcription initiation sites of the hATP1 gene. No TATA box or CAAT box was found near the transcription initiation sites. Comparison with the bovine gene showed that the 5'-flanking region of the hATP1 gene has an unconserved guanine-cytosine (GC) rich region, including several binding motifs of transcriptional factors, such as Sp1, AP-2, and GCF. By functional assay of gene expression, the basal promoter activity was located near the GC rich region. Comparison of the 5'-upstream region of the hATP1 gene with those of the genes for bovine ATP synthase alpha, human beta, and human gamma subunits indicated three common sequences, suggesting that putative cis-elements coordinate the expressions of the three subunit genes for the ATP synthase. The enhancer activities derived from the 5'-deletion mutants of a hATP1-CAT chimeric gene were different in cell lines from four different human tissues, suggesting the existence of cell type-specific gene regulation.