Arachidonic acid metabolites (eicosanoids) have been implicated in mediating actions of cytokines in different tissues. In this study, we tested inhibitors of arachidonate metabolism for possible protection against the toxic effects of the cytokine combination of tumor necrosis factor (TNF, 100 U/ml) and interferon-γ (IFN-γ, 100 U/ml) in rat islet cell monolayer cultures, using a ⁵¹Cr release cytotoxicity assay to measure islet cell lysis (% ⁵¹Cr release). The toxic effect of TNF/IFN-γ (26.6 ± 3.7%) was inhibited partially by both a cyclooxygenase inhibitor, indomethacin and a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), and combination of maximally effective concentrations of Indo and NDGA (30 μM) produced further protection against TNF/IFN-γ-induced lysis (3.5 ± 0.9%). Also, the combined cyclo/lipoxygenase inhibitors, oxyphenbutazone and eicosa 5,8,11,14 tetrynoic acid, as well as the phospholipase A₂ inhibitor, bromophenacyl bromide, significantly inhibited the cytotoxic effect of TNF/IFN-γ. Whereas indomethacin and NDGA did not prevent TNF/IFN-γ-induced inhibition of insulin release, this recovered after cytokine removal from cultures protected by the cyclo/lipoxygenase inhibitors. These results suggest that arachidonate metabolites may be involved in mediating the cytotoxic and not the functional inhibitory effects of TNF and IFN-γ in islet cells. (Endocrinology126: 67–71, 1990)