We have reported recently the isolation of a cDNA for nuclear encoded subunit Vb of mouse cytochrome c oxidase by screening mouse bone marrow and kidney cDNA libraries. In the present study, this cDNA was used as a probe to screen a mouse genomic library and isolate the complete gene encoding subunit Vb. Southern blot hybridization of mouse genomic DNA with the cDNA probe suggested the occurrence of multiple genes including many retroinserts. Restriction analysis followed by Southern blot hybridization of genomic clones was used to identify the putative retroinserts from the intron containing genes. Of the 10 initial genomic clones isolated, one clone (MG3) showing the most complex hybridization pattern was found to contain the complete gene for subunit Vb. The DNA sequence analysis show that the subunit Vb gene contains four exons of 149, 73, 99, and 189 bases interrupted by three relatively small introns of 520, 165, and 648 nucleotides in a gene spanning about 2.5 kilobase pairs. As determined by a combination of primer extension and S1 protection analyses, the major transcription start site appears to be located 49 nucleotides upstream of the translation initiation codon. The ability of the 5' upstream DNA to initiate transcription was studied using the chloramphenicol acetyltransferase (CAT) expression plasmids in NIH 3T3 cells. Using this system we observed that a segment of the gene spanning nucleotides -574 to +45 can drive the transcription of CAT gene in an orientation dependent manner. The upstream region of subunit Vb gene lacks the TATA and CAAT elements, although it contains several GC rich elements and a pyrimidine rich stretch around the transcription start site.