Most mitochondrial proteins are synthesized with cleavable amino-terminal targeting signals that interact with the mitochondrial import machinery to facilitate their import from the cytosol. We previously reported that the presequence of the F1-ATPase β subunit precursor (pre-F1β) acts as an intramolecular chaperone that maintains the precursor in an import-competent conformation prior to import (P. Hajek, J. Y. Koh, L. Jones, and D. M. Bedwell, Mol. Cell. Biol. 17:7169–7177, 1997). We also found that a mutant form of pre-F1β with a minimal targeting signal (Δ1,2 pre-F1β) is inefficiently imported into mitochondria because it rapidly folds into an import-incompetent conformation. We have now analyzed the consequences of reducing the pre-F1β targeting signal to a minimal unit in more detail. We found that Δ1,2 pre-F1β is more dependent upon the Tom70p receptor for import than WT pre-F1β is, resulting in a growth defect on a nonfermentable carbon source at 15°C. Experiments using an in vitro mitochondrial protein import system suggest that Tom70p functions to maintain a precursor containing the Δ1,2 pre-F1β import signal in an import-competent conformation. We also identified PDR3, a transcriptional regulator of the pleiotropic drug resistance network, as a multicopy suppressor of the mitochondrial import defects associated with Δ1,2 pre-F1β in a tom70Δ strain. The overproduction of PDR3 mediated this effect by increasing the import of Δ1,2 pre-F1β into mitochondria. This increased the mitochondrial ATP synthase activity to the extent that growth of the mutant strain was restored under the selective conditions. Analysis of the transcription patterns of components of the mitochondrial outer membrane import machinery demonstrated that PDR3 overproduction increased the expression of TOM72, a little studied TOM70 homologue. These results suggest that Tom72p possesses overlapping functions with Tom70p and that the pleiotropic drug resistance network plays a previously unappreciated role in mitochondrial biogenesis.