Mitochondria contain a small genome that codes for approx 1% of the total number of proteins that reside in the mitochondria. Hence, most mitochondrial proteins are encoded for by the nuclear genome. After transcription in the nucleus these proteins are synthesized by cytosolic ribosomes. Like proteins destined for other organellar compartments, mitochondrially destined proteins possess targeting signals within their primary or secondary structure that direct them to the organelle with the assistance of cytosolic factors. Very specialized and discriminatory protein translocase complexes in the mitochondrial membranes, intermembrane space, and matrix are then engaged for the translocation, sorting, integration, processing, and folding of the newly imported proteins. The principles of protein targeting into mitochondria have been and are still being unraveled, mostly by studies with the yeast Saccharomyces cerevisiae and the fungus Neurospora crassa. In this chapter the major principles of mitochondrial protein targeting as currently understood will be discussed as a foundation for the experimental methods discussed later in this volume.