Two mammalian mitochondrial initiation factors have been identified. Initiation factor 2 (IF2(mt)) selects the initiator tRNA (fMet-tRNA) and promotes its binding to the ribosome. Initiation factor 3 (IF3(mt)) promotes the dissociation of the 55S mitochondrial ribosome into subunits and may play additional, less-well-understood, roles in initiation complex formation. Native bovine IF2(mt) was purified from liver a number of years ago. The yield of this factor is very low making biochemical studies difficult. The cDNA for bovine IF2(mt) was expressed in Escherichia coli under the control of the T7 polymerase promoter in a vector that provides a His(6)-tag at the C-terminus of the expressed protein. This factor was expressed in E. coli and purified by chromatography on Ni-NTA resins. The expressed protein has a number of degradation products in partially purified preparations and this factor is then further purified by high-performance liquid chromatography or gravity chromatography on anion exchange resins. IF3(mt) has never been purified from any mammalian system. However, the cDNA for this protein can be identified in the expressed sequence tag (EST) libraries. The portion of the sequence encoding the region of human IF3(mt) predicted to be present in the mitochondrially imported form of this factor was cloned and expressed in E. coli using a vector that provides a C-terminal His(6)-tag. The tagged factor is partially purified on Ni-NTA resins. However, a major proteolytic fragment arising from a defined cleavage of this protein is present in these preparations. This contaminant can be removed by a single step of high-performance liquid chromatography on a cation exchange resin. Alternatively, the mature form of IF3(mt) can be purified by two sequential passes through a gravity S-Sepharose column.