We have analyzed the mitochondrial RNA of a yeast nuclear pet mutant with no cytochrome oxidase activity. The product of the gene affected in this mutant appears to be necessary for the correct maturation of the mitochondrial pre-mRNA of the cytochrome oxidase subunit I. It does not affect, however, the overall splicing of cytochrome b pre-mRNA or the intron excision of the 21S ribosomal RNA precursor. This gene has been isolated by genetic complementation in yeast, and its DNA sequence has been determined. It is transcribed, as detected by S1 mapping experiments, and could encode a protein of 436 amino acids.