Complete cDNAs encoding human mitochondrial translational initiation factor 2 (IF-2mt) have been obtained from liver, heart, and fetal brain cDNA libraries. These cDNAs have a long open reading frame 2181 residues in length encoding a protein of 727 amino acids. Overall, human IF-2mt has 30-40% identity to the corresponding prokaryotic factors. Surprisingly, it is no more homologous to yeast IF-2mt than to the IF-2s from bacterial sources. The greatest region of conservation lies in the G-domain of this factor with less conservation in the COOH-terminal half of the protein and very little homology near the amino terminus. The 5'-untranslated leaders of the liver and heart cDNAs contain a number of short open reading frames. These sequences may play a role in the translational activity of the IF-2mt mRNA. Northern analysis indicates that the IF-2mt gene is expressed in all tissues but that the level of expression varies over a wide range.