The mammalian mitochondrial genome contains a single tRNAMet gene that gives rise to the initiator and elongator tRNAMet. It is generally believed that mitochondrial protein synthesis begins with formylmethionyl-tRNA, which indicates that the formylation of mitochondrial Met-tRNA specifies its participation in initiation through its interaction with initiation factor 2 (IF-2). However, recent studies in yeast mitochondria, suggest that formylation is not required for protein synthesis. In addition, bovine IF-2mt could replace yeast IF-2mt in strains that lack fMet-tRNA which suggests that this paradigm may extend to mammalian mitochondria. Here, the importance of the formylation of mitochondrial Met-tRNA for the interaction with IF-2mt was investigated by measuring the ability of bovine IF-2mt to bind mitochondrial fMet-tRNA. In direct binding experiments, bovine IF-2mt has a 25-fold greater affinity for mitochondrial fMet-tRNA than Met-tRNA, using either the native mitochondrial tRNAMet or an in vitro transcript of bovine mitochondrial tRNAMet. In addition, IF-2mt will not effectively stimulate mitochondrial Met-tRNA binding to mitochondrial ribosomes, exhibiting a 50-fold preference for fMet-tRNA over Met-tRNA in this assay. Finally, the region of IF-2mt responsible for the interaction with fMet-tRNA was mapped to the C2 sub-domain of domain VI of this factor.