The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is directed to mitochondria by its amino-terminal leader peptide. To define the critical residues and/or regions in the OTC leader peptide, we have synthesized OTC precursors with alterations in the leader portion. Analysis of deletions reveals that the middle portion of the 32 residue leader peptide is absolutely required for both mitochondrial uptake and proteolytic processing, whereas NH2-terminal and penultimate COOH-terminal portions are not. Analysis of precursors with single substitutions revealed complete loss of function when arginine 23 was substituted with glycine. Additional substitutions suggested that the critical role of this arginine residue may be mediated by participation in a local secondary structure, very likely an alpha-helix, which is proposed to be an essential element in the midportion of the leader peptide.