The 13.2-kDa subunit of NADH:ubiquinone oxidoreductase has been shown to be an integral part of the bovine iron-sulfur (IP) part of the protein. This subunit has been shown to interact with at least two other protein subunits of the IP fragment. The amino acid (aa) sequence of this subunit, determined from an acid extract of rat heart was used to generate an oligodeoxyribonucleotide probe which allowed isolation of a cDNA coding for the rat homologue of 13.2-kDa IP. The cDNA was used as a probe of a rat genomic DNA library and two clones were isolated, one of which contained the entire coding region for 13.2-kDa IP. Southern analysis indicates that the IP13 sequence exists as a single copy gene. The sequence of the genomic clone contains one intron and promoter elements including a TATAAA region. The 5' flank region has several potential regulatory sites, most notably regions similar to the nuclear respiratory factor 1 (NRF-1) motif, found in other genes which code for mitochondrial proteins [Evans and Scarpulla, Genes Dev. 4 (1990) 1023-1034]. The core domain of the deduced rat aa sequence has a high degree of identity with the mouse and cow homologues of this protein. The high degree of conservation of this protein indicates that the protein is essential for the function of complex I.