Background.Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects, though the exact mechanism(s) are unknown. This study investigated the effects of eicosapentanenoic acid (EPA), a major component of fish oil, on transcriptional regulation of tumor necrosis factor (TNF) gene in lipopolysaccharide (LPS)-stimulated macrophages (MØ).Methods.RAW 264.7 cells, a mouse MØ cell line, were grown in EPA-rich media for 24–48 h. MØ were washed and exposed toEscherichia coliLPS (1 μg/ml) for 2 h. TNF mRNA expression was measured by Northern blot assays. Total nuclear extracts were harvested for the measurement of NFκB with electrophoretic mobility shift assays. Supershift assays were performed with anti-P50 or anti-P65 antibodies to show components of NFκB dimers. TNF production was determined by L929 bioassays.Results.LPS stimulated RAW cell TNF mRNA expression and NFκB activity. In contrast, RAW cells grown in EPA-rich media had less TNF mRNA expression and an altered composition of the NFκB subunits (P65/P50 dimers) in the presence of LPS. TNF production by LPS-stimulated MØ was reduced by EPA.Conclusions.The inhibitory effect of EPA on LPS-stimulated MØ TNF gene transcription and protein elaboration is, in part, mediated through altering NFκB activation by reducing the P65/P50 dimers.