N-3 polyunsaturated fatty acids (PUFAs) are known to have anti-inflammatory effects. Excess production of nitric oxide (NO) is associated with inflammation. Therefore, we examined the effects of PUFAs on NO production and inducible NO synthase (iNOS) expression by stimulated murine macrophages. One typical n-3 PUFA docosahexaenoic acid (DHA) strongly inhibited NO production and iNOS expression in RAW264 macrophages and mouse peritoneal macrophages in a dose-dependent manner. This inhibition was accompanied by inhibiting the oxidative stress-sensitive transcription factor nuclear factor (NF)-kappaB activation. In stimulated macrophages, intracellular peroxides level was enhanced, but pretreatment of DHA dose-dependently inhibited this enhancement. These results suggest that DHA has an antioxidative effect based on the inhibition of the accumulation of intracellular peroxides, and this inhibition caused the suppression of the activation of NF-kappaB, resulting in the inhibition of NO production and iNOS expression. On the other hand, DHA treatment enhanced the level of intracellular glutathione (GSH), and this enhancement is thought to mediate the activity of DHA because lowering the GSH level by inhibiting GSH biosynthesis reversed the DHA-induced suppression of NO production, NF-kappaB activation, and the accumulation of intracellular peroxides. Our results demonstrate that DHA inhibits NO production in macrophages and this inhibition is, in part, mediated by upregulation of GSH.