A single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae. All fumarase translation products are targeted and processed in mitochondria before distribution. Here we show that targeting of fumarase is coupled to translation and initially involves insertion of the protein across the mitochondrial membranes and processing by the matrix protease. Rapid folding of fumarase may determine its requirement for coupling of its translocation with translation and unique route of distribution. The amino termini of most fumarase molecules are translocated across the mitochondrial membranes and processed. Unlike the in vivo situation where these molecules are released into the cytosol, in vitro they remain externally attached to the mitochondria, thereby positioned for release from the organelle. Our model suggests that fumarase displays a unique mechanism of targeting and distribution, which occurs cotranslationally and involves folding and retrograde movement of the processed protein back through the translocation pore.