Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFNgammaR1 and IFNgammaR2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFNgammaRs in fish. They are widely expressed in tissues, with IFNgammaR1 typically more highly expressed than IFNgammaR2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1beta down regulating IFNgammaR1 expression but up regulating IFNgammaR2 expression. Over-expression of the two receptor chains in RTG-2 cells revealed that the level of IFNgammaR2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gammaIP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFNgammaR may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction.