Binding of precursors to import receptors on the mitochondrial surface is one of the earliest steps of protein import into mitochondria. In yeast, one of these receptors is a 70-kDa outer membrane protein termed Mas70p. Pulse-chase studies with intact yeast cells had indicated that Mas70p accelerates the import of all mitochondrial precursors tested. In contrast, import experiments with isolated mitochondria suggested that Mas70p accelerated import of only a subset of precursors (Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brütsch, H., and Schatz, G. (1990) EMBO J. 9, 3191-3200). To resolve this discrepancy, we have now studied the interaction of Mas70p-deficient and wild-type yeast mitochondria with a precursor (pre-alcohol dehydrogenase III) whose import into isolated mitochondria is not accelerated by Mas70p under the usual assay conditions. Mas70p enhanced binding of pre-alcohol dehydrogenase III to the surface of mitochondria in which the electrochemical potential across the inner membrane had been dissipated by an uncoupler; the bound precursor could be efficiently chased into the mitochondria if the potential was restored. The precursor to cytochrome c1 was also bound to mitochondria in a Mas70p-dependent manner. Mas70p also enhanced the direct import of pre-alcohol dehydrogenase III into isolated mitochondria, provided the precursor was first denatured with urea. Under these conditions, the import rate in vitro was more similar to that in intact cells. Mas70p had no effect on the binding or the import of artificial precursors containing mouse dihydrofolate as the "mature" domain. We conclude that Mas70p is an import receptor for most, if not all authentic mitochondrial precursor proteins, but that its function is not always rate-limiting in import experiments with isolated mitochondria.