Studies in humans and murine disease models have clearly shown dietary fish oil to possess anti-inflammatory properties, apparently mediated by the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To determine the mechanisms by which dietary EPA and DHA modulate mouse T-cell activation, female C57BL/6 mice were fed diets containing either 2% safflower oil (SAF), 2% fish oil (FO), or a 2% purified EPA/DHA ethyl ester mixture for 14 days. Splenic CD4 T cells (∼90% purity) or CD8 T cells (∼85% purity) were incubated with agonists which act at the plasma membrane receptor level [anti(α)-CD3/anti(α)-CD28], the intracellular level (PMA/Ionomycin), or at both the receptor and intracellular levels (αCD3/PMA). CD4 T cells stimulated with αCD3/αCD28 or PMA/Ionomycin proliferated and produced principally IL-2 (i.e. a Th1 phenotype), whereas the proliferation of CD4 T cells stimulated with αCD3/PMA was apparently driven principally by IL-4 (i.e. a Th2 phenotype). The IL-4 driven proliferation of putative Th2 CD4 cells was enhanced by dietary n-3 fatty acids (P = 0·02). Conversely, IL-2 production by αCD3/α CD28-stimulated CD4 T cells was reduced in FO-fed animals (P < 0·0001). The αCD3/αCD28-stimulated CD8 cells cultured from FO-fed animals exhibited a significant decrease (P < 0·05) in proliferation. There were no dietary effects seen in αCD3/PMA-stimulated CD8 cells, which produced both IL-2 and IL-4, or in PMA/Ionomycin-stimulated CD8 cells, which produced principally IL-2. These data suggest that dietary n-3 fatty acids down-regulated IL-2 driven CD4 and CD8 activation, while up-regulating the activation of the Th2 CD4 T-cell subset. Thus, the anti-inflammatory effects of n-3 fatty acids may result in both the direct suppression of IL-2-induced Th1 cell activation and the indirect suppression of Th1 cells by the enhanced cross-regulatory function of Th2 cells.