To better understand the early events of the mitochondrial protein import process, we purified the precursor of the F1-ATPase β subunit (pre-F1β) and examined its import into isolated mitochondria. Import of purified urea- denatured pre-F1β did not require cytosolic factors. However, the period of productive import was prolonged by the addition of reticulocyte lysate, suggesting that cytosolic factors such as molecular chaperones were acting to extend the period of import competence of pre-F1β. Purified pre-F1β bound extensively to both cardiolipin-containing liposomes and to intact mitochondria, indicating that a direct interaction between mitochondrial precursors and the mitochondrial outer membrane surface can occur. The ability to chase this surface-bound pre-F1β into mitochondria suggests that precursors bound to the mitochondrial surface can be maintained in an import competent conformation. Finally, our defined mitochondrial import system was used to characterize the ATP requirements of pre-F1β import in the absence of cytosol. We found a strong requirement for ATP on both sides of the mitochondrial inner membrane, suggesting that one or more previously undetected mitochondrial proteins outside the inner membrane utilize ATP to promote efficient pre-F1β import.