All of the mitochondrial tRNAs of Trypanosoma brucei have been shown to be encoded in the nucleus and must be imported into the mitochondrion. The import of nuclearly encoded tRNAs into the mitochondrion has been demonstrated in a variety of organisms and is essential for proper function in the mitochondrion. An in vitro import assay has been developed to study the pathway of tRNA import in T. brucei. The in vitro system utilizes crude isolated trypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleus-encoded tRNA gene cluster. The substrate, composed of tRNA(Ser) and tRNA(Leu), is transcribed in tandem with a 59-nucleotide intergenic region. The tandem tRNA substrate is imported rapidly, while the mature-size tRNA(Leu) fails to be imported in this system. These results suggest that the preferred substrate for tRNA import into trypanosome mitochondria is a precursor molecule composed of tandemly linked tRNAs. Import of the tandem tRNA substrate requires (i) a protein component that is associated with the surface of the mitochondrion, (ii) ATP pools both outside and within the mitochondrion, and (iii) a membrane potential. Dissipation of the proton gradient across the inner mitochondrial membrane by treatment with an uncoupling agent inhibits import of the tandem tRNA substrate. Characterization of the import requirements indicates that mitochondrial RNA import proceeds by a pathway including a protein component associated with the outer mitochondrial membrane, ATP-dependent steps, and a mitochondrial membrane potential.