In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).