It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1, a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex in, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (Δ68–80) was still functional in the cytochrome c1 -deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (Δ168–179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex HI. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171→Asn170Asn171 or Asp170Asp171→Asn170Lys171) made the cytochrome Ci-deficient cells respiration-competent. On the other hand, mutant cytochrome d with the deletion of the first acidic region (Δ68–80) or altered charge distribution in the second region (AspI70Asp171→Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1, but also the QCR6 protein. These results indicate that the acidic regions in cytochrome c1 are essential for the ubiquinol-cytochrome c reductase activity in yeast cells in the absence of the QCR6 protein, and suggest the acidic regions of cytochrome c1 may promote binding of complex III to cytochrome c in cooperation with the QCR6 protein.