In squid (Loligo breekeri) mitochondria, AUA codons are translated as methionine instead of the universal isoleucine. Here, we present the nucleotide sequence of squid mitochondrial tRNA(Met)CAU. This tRNA(Met)CAU has 5-formylcytidine (f5C) at the wobble position of the anticodon, though it is partially modified. This result indicates the common feature with bovine and nematoda mitochondrial systems in that f5C at the wobble position of the anticodon is very likely involved in translation of AUA codons as methionine in squid mitochondria.