: We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA(Glu), tRNA(Thr), and tRNA(Pro)) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome.