Yeast mutants modifying the C-terminal region of mitochondrial cytochrome b were isolated and characterized. A nonsense mutation of the leucine codon 335 (TTA-->TAA), 50 residues before the normal C-terminus, blocks incorporation of heme into the apocytochrome b and prevents growth on non-fermentable substrates. The same defects were observed in a frameshift mutant (after codon 348, TAT-->TATT) in which the last 37 C-terminal residues are predicted to be replaced by a novel sequence of 33 amino acids. Function was regained in the nonsense mutant only by true back mutations restoring a protein of the wild-type sequence. The respiratory capacity was restored to wild-type levels in the frameshift mutant by a variety of single base subtractions located within a window of 24 bases before or after the original +T addition, these pseudo-reversions resulted in single or multiple (up to five) consecutive amino acid replacements between positions 346 and 354 and restored the wild-type sequence from position 355 to 385. These data, combined with hydropathy calculations and sequence comparisons, suggest that the C-terminal domain of cytochrome b forms a transmembrane segment essential for the correct assembly of the cytochrome bc1 complex.