Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator and has also the potential as a prominent biomarker of innate immunity. In this study, we identified and characterized TNF-alpha from bluefin tuna, which is an important cultured species. Two types of TNF-alpha were also cloned incidentally (TNF1 and TNF2). The open reading frame of TNF1 and TNF2 cDNA encoded 247 and 245 amino acids, respectively. The amino acid sequence identity among sea perch, red sea bream, and tiger puffer was 73, 70, 59% for TNF1 and 49, 51, 45% for TNF2, respectively. The identity between TNF1 and TNF2 amino acid sequences of the bluefin tuna was only 43%. The positions of cysteine residues, transmembrane sequence, and protease cleavage site in bluefin tuna TNFs were similar with other reported fish and mammalian TNF-alpha. In a phylogenetic analysis, TNF1 is grouped with other reported Perciformes TNF-alpha. On the other hand, TNF2 is grouped with ayu TNF and is quite distant from the fish TNF-alpha group and lymphotoxin-beta group. While TNF1 mRNA showed no significant difference in all tissues, TNF2 mRNA was expressed significantly higher in the blood than in the gill, intestine, head kidney, spleen, heart, and ovary. In peripheral blood leucocytes (PBL), expressions of TNF2 mRNA were significantly increased by stimulation with lipopolysaccharide, phytohemagglutinin, concanavalin A, pokeweed mitogen, phorbol myristate acetate in vitro, but those of TNF1 were not. Recombinant mature TNF1 and TNF2 proteins significantly enhanced phagocytic activity of PBL. Our results suggest that bluefin tuna possess two types of TNF-alpha homologue, and TNF2 is a potential biomarker for innate immunity.