Post-translational maturation of soluble cytochrome c includes translocation of the precursor polypeptide and heme through the cytoplasmic membrane, proteolytic cleavage of the signal sequence, and covalent attachment of heme. Specific genes for cytochrome c maturation (ccmABCDEFGH in Escherichia coli) are required for holocytochrome c formation, among them genes encoding an ABC transporter (ccmABC). We investigated the requirements of apocytochrome translocation to the periplasm and characterized specific intermediates of the cytochrome c maturation pathway. Apocytochrome precursor was present in the membrane fraction. Translocation of the polypeptide was independent of ccm gene products, but dependent on a functional secretion machinery, as shown by accumulation of preapocytochrome c in the membranes of secA and secY mutants. After translocation, cleavage of the signal sequence allowed the release of apocytochrome into the periplasm, where heme was bound in a ccm-dependent manner. By contrast, non-cleaved holocytochrome c containing covalently bound heme accumulated in the membranes of a lepB mutant, which indicated that signal sequence cleavage and heme attachment are independent steps in the cytochrome c maturation pathway.