ABSTRACT In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides , were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c -type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c -type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c -type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c -type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or β-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.