It is known that each aminoacyl-tRNA synthetase (aaRS) specifically recognizes its cognate tRNAs to ensure the correct translation of the genetic information. However, we had previously demonstrated that mammalian mitochondrial seryl-tRNA synthetase (mt SerRS) can markedly misacylate mitochondrial (mt) tRNA(Gln). To investigate extensively misacylation reactions in mammalian mitochondrion, we purified overall twenty-two mt tRNAs from bovine liver, and determined their misacylation activities using mt SerRS. In addition to tRNA(Gln), tRNA(Ala) and tRNA(Asn) showed weak but significant serylation activities, which raises the possibility that each mammalian mt aaRS can misacylate several non-cognate mt tRNAs in varying degrees, but translational fidelity might be maintained by kinetic discrimination of tRNAs in the network of aaRSs.