Previous work has shown that when yeast mitochondria are incubated in the presence of the presequence peptide pL4(1-22), the peptide is imported and accumulates within the mitochondrial membranes, presumably at the import sites. If the extramitochondrial concentration of peptide is sufficiently high, enough peptide accumulates within the import sites to prevent the uptake of authentic precursor proteins. We have used chemical cross-linking to probe the interaction of this peptide with yeast mitochondrial proteins. We found that radiolabeled pL4(1-22) could be reproducibly cross-linked to a number of polypeptides. Interestingly, nearly all were membrane proteins. Several of the cross-linked proteins were located in the outer membrane, while others were located in the inner membrane. The interaction between the peptide and many of the cross-linked products was shown to be specific by two independent criteria. First, an excess of unlabeled peptide acted as a competitor in the cross-linking reaction, and, second, treatment of the peptide with the alkylating agent N-ethylmaleimide dramatically reduced its ability to form cross-links. Two of the cross-linked species corresponded to the outer membrane proteins, Mas70p and ISP42. Significantly, both of these proteins have previously been shown to play critical roles in mitochondrial protein import. While the role of the other cross-linked proteins in the import process remains to be determined, the results of this study demonstrate that our experimental approach may be useful in identifying components of the import machinery as well as proteins that interact with mitochondrial presequences.