Vertebrate mitochondria use stop codons UAA and UAG decoded by the release factor (RF) MTRF1L and two reassigned arginine codons, AGA and AGG. A second highly conserved RF-like factor, MTRF1, which evolved from a gene duplication of an ancestral mitochondrial RF1 and not a RF2, is a good candidate for recognizing the nonstandard codons. MTRF1 differs from other RFs by having insertions in the two external loops important for stop codon recognition (tip of helix alpha5 and recognition loop) and by having key substitutions that are involved in stop codon interactions in eubacterial RF/ribosome structures. These changes may allow recognition of the larger purine base in the first position of AGA/G and, uniquely for RFs, only of G at position 2. In contrast, residues that support A and G recognition in the third position in RF1 are conserved as would be required for recognition of AGA and AGG. Since an assay with vertebrate mitochondrial ribosomes has not been established, we modified Escherichia coli RF1 at the helix alpha5 and recognition loop regions to mimic MTRF1. There was loss of peptidyl-tRNA hydrolysis activity with standard stop codons beginning with U (e.g., UAG), but a gain of activity with codons beginning with A (AAG in particular). A lower level of activity with AGA could be enhanced by solvent modification. These observations imply that MTRF1 has the characteristics to recognize A as the first base of a stop codon as would be required to decode the nonstandard codons AGA and AGG.