Fish oil (FO) is considered antiinflammatory, but evidence regarding its effect on human cytokine production is conflicting. High linoleic acid (LA) intake may impair any effects of FO. The aim of this study was to investigate how FO combined with high or low LA intake affected ex vivo cytokine production from cultures of whole blood, peripheral blood mononuclear cells (PBMC), and monocytes in healthy men. The study was a double-blinded, controlled, 2 x 2 factorial 8-wk intervention. Sixty-four healthy men were randomized to 5 mL/d FO or olive oil (OO) provided in capsules and to spreads and oils with high or low LA content, resulting in LA intakes of 7 +/- 2% and 4 +/- 1% energy, respectively. We measured eicosapentaenoic acid (EPA) in PBMC and stimulated cytokine production in whole blood and PBMC 24-h cultures before and immediately after intervention and after an 8-wk wash-out period, and in monocyte cultures immediately after intervention. PBMC-EPA was markedly increased by FO (P < 0.001). LA intake did not modify the incorporation of FO and tended to have only a slight effect on PBMC-EPA by itself (P = 0.06). Lipopolysaccharide (LPS)-stimulated whole-blood interleukin (IL)-6 production immediately after intervention was lower with FO than OO (P = 0.02) but did not correlate with PBMC-EPA in the FO groups (r = -0.12; P = 0.53; n = 31). The LA intake did not modify IL-6 production or the effect of FO. Neither FO nor LA intake affected the production of tumor necrosis factor-alpha, IL-10, or interferon-gamma in any of the cultures. In conclusion, FO intake reduced IL-6 production from LPS-stimulated whole blood in healthy men compared with OO, but the effect was not modified by the LA intake.