Respiratory defective mutants of Saccharomyces cerevisiae assigned to complementation group G28 display a deficiency, in the respiratory chain complex coenzyme QH2-cytochrome c reductase. The mutants define a new nuclear gene, designated CBP3, required for the assembly of the complex. Mutations in CBP3 are expressed in the absence of spectrally and immunologically detectable cytochrome b, a catalytic subunit of coenzyme QH2-cytochrome c reductase. The mutational block responsible for the cytochrome b deficiency has been ascribed to a post-translational step based on the observation that cbp3 mutants have wild type concentrations of cytochrome b mRNA and are capable of synthesizing the apoprotein. Western analysis has revealed that cbp3 mutants have reduced levels of a subset of subunit polypeptides of the coenzyme QH2-cytochrome c reductase complex that include apocytochrome b, the iron-sulfur protein, core 4 (14-kDa subunit), and core 5 (11-kDa subunit). A similar phenotype has previously been reported in strains that fail to assemble the complex as a result of mutations in the noncatalytic core subunits. The CBP3 gene has been cloned by transformation of a mutant from complementation group G28 with a yeast genomic library. The gene is 1005 nucleotides long and codes for a primary translation product of 39 kDa. A transcript of a size commensurate with the length of the CBP3 reading frame is detected in total and poly(A+)-enriched RNA. The amino-terminal region of the CBP3 product is basic and probably corresponds to a cleavable mitochondrial targeting signal. An antibody obtained against a trpE/CBP3 fusion protein detects a protein of 40 kDa in wild type yeast mitochondria. This protein is absent in a mutant construct containing a partially deleted copy of the gene. The CBP3 protein is a membrane constituent, although attempts to demonstrate its physical association with the other subunits of coenzyme QH2-cytochrome c reductase have been unsuccessful.