The mammalian mitochondrial methionyl-tRNA transformylase (MTF mt ) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTF mt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTF mt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTF mt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTF mt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNA fMet ) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNA mMet ) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTF mt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator MettRNAs. These observations are discussed in terms of the presence of a single tRNA Met gene in mammalian mitochondria.