Elongation factor Tu (EF-Tu) is responsible for the delivery of the aminoacyl-tRNAs (aa-tRNA) to the ribosome during protein synthesis. The primary sequence of domain II of EF-Tu is highly conserved. However, several residues thought to be important for aa-tRNA binding in this domain are not conserved between the mammalian mitochondrial and bacterial factors. One of these residues is located at position 290 (Escherichia coli numbering). Residue 290 is Gln in most of the prokaryotic factors but is conserved as Leu (L338) in the mammalian mitochondrial factors. This residue is in a loop contacting the switch II region of domain I in the GTP-bound structure. It also helps to form the binding pocket for the 5' end of the aa-tRNA in the ternary complex. In the present work, Leu338 was mutated to Gln (L338Q) in EF-Tu(mt). The complementary mutation was created at the equivalent position in E. coli EF-Tu (Q290L). EF-Tu(mt) L338Q functions as effectively as wild-type EF-Tu(mt) in poly(U)-directed polymerization with both prokaryotic and mitochondrial substrates and in ternary complex formation assays with E. coli aa-tRNA. However, the L338Q mitochondrial variant has a reduced affinity for mitochondrial Phe-tRNA(Phe). E. coli EF-Tu Q290L is more active in poly(U)-directed polymerization with both mitochondrial and prokaryotic substrates and has a higher GTPase activity in both the absence and presence of ribosomes. Surprisingly, while E. coli EF-Tu Q290L is more active in polymerization with mitochondrial Phe-tRNA(Phe), this variant has low activity in the formation of a stable ternary complex with mitochondrial aa-tRNA.