We have isolated two overlapping clones covering the entire length of the gene of nuclear-encoded sub-unit IV of cytochrome c oxidase (COXIV) from a rat genomic library in Charon 4A and determined the structural organization of the gene. The gene spans approximately 7.0 kilobases and contains five exons interrupted by four introns. Of these exons, exon 2 codes for a whole length of the presequence of the rat COXIV precursor protein, while exons 3 to 5 encode three distinct structural domains of the mature protein. The 5'-flanking region of the gene lacks conventional TATA and CAAT boxes, but has a high G + C content and contains two putative binding sites for transcription factor SP1 and a sequence resembling the AP-4 responsive element. These results indicate that the promoter region of the rat COXIV gene possesses characteristic features common in housekeeping genes. The chloramphenicol acetyltransferase assay performed by constructing an improved phagemid, pBlueCAT3, revealed that a 773-base pair DNA fragment immediately preceding the cap site has a strong promoter activity. An octanucleotide sequence, -TTCTTGGT-, which is very close to the yeast HAP2/HAP3 responsive element, is located in the 5'-upstream region of the present gene.