Apocytochrome c follows a unique pathway into mitochondria. Import does not require the general protein translocation machinery, protease-sensitive components of the outer membrane, or a membrane potential across the inner membrane. We investigated the membrane binding and translocation steps of the import reaction using purified outer membrane vesicles (OMV) from Neurospora crassa mitochondria. OMV specifically bound, but did not import apocytochrome c. If, however, specific antibodies were enclosed inside OMV, apocytochrome c was accumulated in soluble form in the lumen. Import was reversible, since apocytochrome c became accessible to external protease after release from the antibodies. Thus, OMV are competent of translocating apocytochrome c into their lumen, but lack a binding partner which traps the apoprotein. In intact mitochondria, cytochrome c heme lyase (CCHL), a peripheral protein of the inner membrane, serves such a function by stably associating with apocytochrome c in a complex which is detectable by co-immunoprecipitation. We suggest a model for the import mechanism of apocytochrome c in which the apoprotein specifically associates with and reversibly passes across the outer membrane. Translocation is rendered unidirectional by stable association with CCHL which serves as a "trans side receptor." Finally, heme is attached by CCHL and the holoprotein folds into its native structure.