We identified and characterized two isoforms of tumor necrosis factor-alpha (TNFalpha) from the goldfish, TNFalpha-1 and TNFalpha-2. At the protein level, goldfish TNFalpha-1 and TNFalpha-2 were most homologous to carp TNFalpha-1 and TNFalpha-2, respectively. Phylogenetically, the two goldfish isoforms grouped most closely with the carp TNFalpha isoforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of goldfish TNFalpha-1 and TNFalpha-2 in all tissues with TNFalpha-2 mRNA levels higher than TNFalpha-1 in all tissues examined. A modest up-regulation in expressions of goldfish TNFalpha-1 and TNFalpha-2 in kidney-derived monocytes and significant increase in expression of both isoforms in mature macrophages were observed in response to activation with macrophage-activating factors. TNFalpha-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNFalpha-2) was functionally characterized. The rTNFalpha-2 induced a dose-dependent chemotactic response and enhanced phagocytosis of primary goldfish macrophages. Furthermore, rTNFalpha-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary goldfish macrophages. Our results indicate that goldfish TNFalpha is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the goldfish.