Imidazoquinolines represented by imiquimod and its derivative S-27609, have previously been shown to have potent antiviral and antitumor activity in several mammalian models. Much of the antiviral properties of imidazoquinolines have been ascribed to induction of IFN-alpha in peripheral blood mononuclear cells most notably plasmacytoid dendritic cells. Toll-like receptor (TLR) 7 has been identified as the receptor for imiquimod and S-27609 in mammals. In this work we show that S-27609 induces expression of IFN-alpha1/alpha2, Mx, ISG15 and IFN-gamma in organs of Atlantic salmon, which suggests that salmon responds to S-27609 through a TLR7-like receptor. The kinetics of gene expression in liver and head kidney induced by S-27609 was compared with that induced by the double-stranded RNA poly I:C, which is a ligand for TLR3 and the RNA helicase MDA5. In liver, S-27609 and poly I:C both induced transcripts for IFN-alpha1/alpha2, Mx and ISG15 with a peak at about 24h. In head kidney, poly I:C induced IFN-alpha1/alpha2 and Mx with one peak at about 24h. In contrast, S-27609 induced a biphasic increase of IFN-alpha1/alpha2 and Mx transcripts in head kidney with a minor peak at 14-24h and another increase starting at 72h. The other major difference in gene induction by the two stimulants was that S-27609 induced much lower levels of IFN-alpha1/alpha2 than poly I:C during the early time stages (14-48h) both in liver and head kidney. The difference in induction of IFN-alpha1/alpha2 by S-27609 and poly I:C may be a consequence of different cell targets for the two stimulants where S-27609 primarily induces IFNs through immune cells whereas poly I:C induces IFNs in most nucleated cells.