Cytochrome c oxidase (COX) contains ten nuclear encoded subunits, three of them known to show tissue isoforms in mammals. We have now found a fourth isoform, for subunit IV, in human, rat and mouse (COX IV-2). Comparison of the two human isoform genes shows a similar structural organization, including an overall size of about 8 kb, the presence of five exons, and the initiation of translation in the second exon, consistent with formation by gene duplication. Also consistent is the higher identity of precursor peptides of 78% within the new IV-2 isoform (average in the three species) compared to 44% average identity with the IV-1 isoform. Northern analysis and quantitative PCR with human and rat tissues show high IV-2 expression in adult lung and lower expression in all other tissues investigated, including fetal lung. In contrast, the IV-1 isoform is ubiquitously expressed. In situ hybridizations were performed to localize isoform transcripts in rat lung. Both isoforms are found in similar ratios in most lung cell types except for smooth muscle and respiratory epithelium, which have a IV-2 and a IV-1 preference, respectively. Structural modeling of the IV-2 isoform from human, based on the bovine crystal data, produces a conformation in which two of three conserved cysteine groups, exclusively present in the mammalian IV-2 isoform, are in close proximity. The formation of a cysteine bond and the implications for function of these sequence differences for subunit IV, which plays a pivotal role in COX regulation, are discussed.