Import of most nucleus-encoded preproteins into mitochondria is mediated by N-terminal presequences and requires a membrane potential and ATP hydrolysis. Little is known about the chemical nature and localization of other mitochondrial targeting signals or of the mechanisms by which they facilitate membrane passage. Mitochondrial heme lyases lack N-terminal targeting information. These proteins are localized in the intermembrane space and are essential for the covalent attachment of heme to c type cytochromes. For import of heme lyases, the translocase of the mitochondrial outer membrane complex is both necessary and sufficient. Here, we report the identification of the targeting signal of mitochondrial heme lyases in the third quarter of these proteins. The targeting sequence is highly conserved among all known heme lyases. Its chemical character is hydrophilic because of a large fraction of both positively and negatively charged amino acid residues. These features clearly distinguish this signal from classical presequences. When inserted into a cytosolic protein, the targeting sequence directs the fusion protein into the intermembrane space, even in the absence of a membrane potential or ATP hydrolysis. The heme lyase targeting sequence represents the first topogenic signal for energy-independent transport into the intermembrane space and harbors two types of information. It assures accurate recognition and translocation by the translocase of the mitochondrial outer membrane complex, and it is responsible for driving the import reaction by undergoing high-affinity interactions with components of the intermembrane space.