Most mitochondrial proteins are encoded in the nucleus and translated in the cytoplasm as larger precursors containing NH2-terminal "leader" peptides, which are strikingly basic in overall amino acid composition. Recent experiments indicate that these leader peptides are both necessary and sufficient to direct post-translational recognition and import of precursors by mitochondria. In this report, we demonstrate a critical role for one or more of the basic arginine residues in the leader peptide of the subunit precursor for the human mitochondrial matrix enzyme, ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3). The distal three of four basic residues, all arginines, in the leader peptide of ornithine transcarbamoylase were replaced at once with charge-neutral glycine residues. The altered ornithine transcarbamoylase precursor failed to be taken up by intact mitochondria in vitro. Moreover, it also failed to be proteolytically cleaved upon incubation with a mitochondrial matrix fraction containing the Zn2+-dependent protease, which normally cleaves the leader peptide.