The protonmotive cytochrome b protein of the mitochondrial bc1 respiratory chain complex contains two reactions centers, designated Qo and Qi, which can be distinguished by the effects of different inhibitors. The nucleotide sequences have been determined of the mitochondrial cytochrome b genes from a series of mouse cell mutants selected for increased inhibitor resistance. Each mutant contains a single nucleotide change which results in an amino acid substitution. When the proximity of the altered amino acid residues to the histidines involved in heme ligation is considered, the results support a model for cytochrome b folding in which there are eight transmembrane domains rather than the nine of the Widger-Saraste model. Replacement of the Gly38 residue by valine results in resistance to the Qi inhibitors antimycin A and funiculosin but not 2-n-heptyl-hydroxyquinoline-N-oxide. Based upon sequence comparisons of mitochondrial and bacterial cytochrome b and chloroplast b6 proteins, the region of the molecule involved in antimycin binding is as highly conserved as those domains involved in heme ligation. It is suggested that the antimycin binding domain of cytochrome b is involved in forming the Qi reaction center. Alterations of the Gly142 and Thr147 residues result in resistance to myxothiazol and stimatellin, respectively. While both inhibitors block the Qo reaction center, the two mutations do not confer cross-resistance to each other. This region of cytochrome b is the most highly conserved during evolution and these inhibitor binding sites probably occur within the protein domain constituting the Qo reaction center. In addition, there is a less conserved region of the protein, defined by the Leu294 residue, which may function in binding the hydrophobic portions of Qo inhibitors.