The Saccharomyces cerevisiae COX5b gene contains a small intron that is unique in two respects. First, it interrupts the ATG codon that initiates translation of the COX5b product. Second, it contains a sequence at the 5' splice junction (5'-GCATGT-3') that differs from the highly conserved yeast hexanucleotide (5'-GTAPyGT-3') and from the 5'-GT found at the corresponding position in nearly all introns of eucaryotic protein-coding genes. We have analyzed both the transcripts derived from the COX5b gene and the splicing of its intron. We show here that an unspliced mRNA precursor constituted a minor fraction of the total COX5b message, even when the gene was overexpressed. We also show that both major transcripts derived from COX5b had been spliced. Our results suggest that at least in the case of COX5b, a 5'-GC can function as efficiently as the highly conserved 5'-GT in the splicing reaction.