The nucleotide sequence of the cytochrome oxidase subunit 2 (COX2) gene has been obtained from cloned mitochondrial DNA segments of Neurospora crassa. The coding sequences have been identified on the basis of protein sequence homology with the subunit 2 of cytochrome oxidase from yeast and man. The postulated precursor of the N. crassa subunit 2 protein is 250 amino acids long, with a molecular weight of 28,700. As in the tRNA and rRNA genes, the subunit 2 gene is flanked by G + C-rich palindromic sequences, which are highly conserved in N. crassa mitochondria. Three major transcripts have been detected by Northern blot hybridization. A transcript of 1100 bases is tentatively considered the fully processed mRNA. Furthermore, S1 nuclease protection experiments have revealed that the putative subunit 2 mRNA has a 330 nucleotide long 5' leader sequence.