A nuclear gene (CBP1) of Saccharomyces cerevisiae involved in 5' end processing of the cytochrome b pre-mRNA has been cloned and sequenced. The gene was originally selected by transformation of cbp1 mutants with a recombinant plasmid bank of random fragments of yeast nuclear DNA ligated to a plasmid vector with autonomous replicative function in yeast. The recombinant plasmid pG60/T10 with a nuclear DNA insert of 6.7 kilobase pairs (kb) was used to construct a new plasmid pool with the CBP1 gene on smaller fragments of nuclear DNA. A number of subclones have been isolated with plasmids carrying inserts of 2.4 to 6.1 kb. The plasmid designated pG60/T31 confers respiratory competency to cbp1 mutants and restores their ability to synthesize mature cytochrome b mRNA. The pG60/T31 plasmid has a nuclear DNA insert of 2.4 kb. The sequence of the cloned fragment reveals only one open reading frame capable of coding for a protein. The reading frame is 1962 nucleotides long and codes for a basic polypeptide with a molecular weight of 76,140. A transcript of a size commensurate with the length of the gene has been detected in wild type yeast.