The homoacetogenic anaerobic bacterium Sporomusa sphaeroides was mutagenized with UV light. Taking advantage of the ampicillin enrichment technique and a newly developed test for the detection of heme in bacterial colonies, the cytochrome-deficient mutant strain S. sphaeroides BK824 was isolated. In contrast to the wild type, this mutant strain failed to grow on betaine, betaine plus methanol, H2 plus CO2, and methanol plus CO2. Growth on betaine plus formate, betaine plus H2, betaine plus pyruvate, methanol plus H2 and CO2, and acetoin was not impaired. All enzymes of the Wood pathway as well as hydrogenase and carbon monoxide dehydrogenase were detectable at comparable activities in both the wild type and the cytochrome-deficient mutant. Labeling experiments with [14C]methanol demonstrated the inability of S. sphaeroides BK824 to oxidize methyl groups. The role of cytochromes in electron transport steps associated with the Wood pathway enzymes and their possible role in energy conservation during autotrophic growth in acetogens are discussed.