We have characterized two yeast mutants deficient in the splicing of transcripts of the mitochondrial gene for cytochrome c oxidase subunit I (coxI). Both map to the first intron (aI1). RNA blot analysis shows that in addition to a reduced (mutant M15-190) or blocked (mutant M12-193) excision of the mutated intron aI1, the mutants are unable to excise the adjacent aI2 intron, the reading frame of which displays an amino acid sequence similarity to aI1. Splicing of the downstream introns is not affected, however. Sequence analysis of the first mutant DNA (M12-193) reveals a premature termination of the intron-encoded open reading frame, followed by two alterations at a short distance downstream. The other (M15-190) contains 11 separate changes. Although these occur in the intron reading frame, their main effect on RNA splicing may be exerted through the disturbance of intron secondary structure proposed for the 5' end of several group II introns. The implications of these findings in relation to maturase function and structure of intron aI1 are discussed.