A homologous in vitro poly(U)-directed translation system has been established using animal mitochondrial ribosomes, elongation factors (EF) and phenylalanyl-tRNA(Phe). The rate of incorporation of phenylalanine into polyphenylalanine in the mitochondrial system is slower than that observed for the homologous Escherichia coli system. E. coli ribosomes can be used in place of mitochondrial ribosomes in this system with only a slight decrease in the efficiency of phenylalanine incorporation from mitochondrial Phe-tRNA. However, E. coli elongation factor Tu (EF-Tu) cannot replace the mitochondrial EF-Tu in promoting the use of mitochondrial Phe-tRNA. The interaction between EF-Tu and mitochondrial Phe-tRNA was investigated by using the ability of EF-Tu to protect the aminoacyl-tRNA bond from hydrolysis. These results showed that both mitochondrial and E. coli EF-Tus are capable of interacting with mitochondrial Phe-tRNA. However, ribosomal A-site binding assays demonstrated that efficient binding of the mitochondrial Phe-tRNA to the ribosomal A-site was only obtained with the homologous mitochondrial EF-Tu.