The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRNA Met , whereas the only other trypanosomal tRNA Met , the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRNAs Met carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA Met is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA Met becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA Met is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA Met formyltransferases, it has about twice the mass of any of these proteins.