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In this study, genes of two distinct tissue inhibitors of metalloproteinases-2 (TIMP-2) from Japanese puffer fish Fugu rubripes, Fugu TIMP-2a and TIMP-2b, were cloned. The open reading frames of Fugu TIMP-2a and TIMP-2b cDNAs are composed of 660 and 657 nucleotides and 220 and 219 amino acids, respectively. Both Fugu TIMP-2s contain 12 cysteine residues, whichmight form six disulfide bonds as in other animals TIMP-2s. Reverse-transcribed polymerase chain reaction analysis showed the mRNAs of Fugu TIMP-2a and TIMP-2b to be expressed in some tissues examined with different expression patterns. These findings suggest that the two distinct Fugu TIMP-2s might perform different functions in Fugu tissues.