We have isolated two non-overlapping clones containing the genes for subunit Vb of cytochrome c oxidase (COXVb) from a rat genomic library in Charon 4A using a newly isolated full-length cDNA as a probe. One of the two genomic clones, designated as lambda COXVb741, contained a functional gene (COXVb-1), while the other one, designated as lambda COXVb211, contained an intronless processed pseudogene (COXVb-2). The COXVb-1 gene spans approximately 1.8 kb and consists of four exons interrupted by three introns. The nucleotide sequences of all exons are completely identical to the corresponding sequences of the rat liver and brain COXVb cDNAs, indicating that this gene is actually expressed. The 5'-flanking region of the gene lacks conventional TATA and CAAT boxes, but exhibits strong promoter activity in the chloramphenicol acetyltransferase (CAT) assay. Deletional analysis and gel shift assay of the 5'-flanking region suggested that the binding of nuclear factor Sp-1 could be essential for transcription of the gene. Southern blotting analysis implied the occurrence of multiple COXVb genes in the rat genome. However, the results of the present experiments suggest that only the COXVb-1 gene is expressed in rat tissues.