An extragenic suppressor mutation, mim2-1, which compensates yeast mitochondrial mutants deficient in splicing of the cytochrome b gene, has been mapped and sequenced. The mutation is due to a single G leads to A transition in the long open reading frame of the fourth intron of the oxidase subunit one gene. It causes the replacement of a glutamic codon by a lysine codon and the expression of a novel mRNA maturase active in splicing. Evolution and regulatory connections between homologous introns of nonhomologous genes are discussed.