In Saccharomyces cerevisiae, the mitochondrial gene encoding the subunit I of cytochrome c oxidase (oxi-3 gene) is interrupted by intervening sequences. In this report, a nuclear mutation [referred to as mss51 in Faye, G. & Simon, M. (1983) Cell 32, 77-87] that specifically affects the processing of oxi-3 pre-mRNA was further characterized. DNA probes covering each oxi-3 exon-intron boundary were individually hybridized to wild-type and mutant mitochondrial RNA. By a technique relying on the S1 nuclease resistance or sensitivity of the RNA X DNA hybrids thereof, we have shown which site needs the MSS51 gene product to be cleaved. The mutation in the MSS51 gene gave rise to a complex pattern of splicing: the third intron was excised efficiently but the first two introns remained bracketed by their flanking exons. Further, the fourth and fifth introns were only partially split from their common exon and remained fused to their upstream and downstream flanking exon, respectively. Several plausible roles for the MSS51 gene product are discussed.