The bovine gene for the nuclear-encoded heart/muscle isoform of cytochrome c oxidase subunit VIa (COX6A1) was isolated from a library of bovine genomic DNA in lambda EMBL3 and sequenced. The gene spans 760 bp and comprises three exons and two small introns. Exon 1 encodes a 193 bp 5' untranslated region, a 12 amino acid presequence, and the first 12 amino acids of the mature COX VIa protein. Exon 2 encodes amino acids 13 to 58, and exon 3 amino acids 59 to 85 plus the 35 bp 3' untranslated region. Exons 2 and 3 are separated by a small intron of only 96 bp. All exon-intron boundaries matched the consensus splice junction sequences. COX6A1 transcripts are present in RNA from bovine heart but not brain. Primer extension and ribonuclease protection assays were used to map the 5' ends of COX6A1 transcripts in heart; both methods identified several clusters of transcription initiation sites, indicating that COX6A1 mRNA is heterogeneous at the 5' end. The proximal 5' flanking region is AT-rich and contains potential basal promoter elements, such as TATA and CCAAT boxes, associated with tissue-specific genes. A single consensus binding site for the muscle-specific transcription factor, MyoD1, was also located within this AT-rich region. The distal promoter region contained a perfect AP4 site plus potential binding sites for enhancer elements (NRF-1, Mt1, Mt3, and Mt4) proposed to regulate expression of genes for mitochondrial proteins.