Phosphorylation of extracellular signal-regulated kinases (ERK1/ERK2) has been implicated in cell proliferation of mammalian cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1/ERK2 phosphorylation, stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGFalpha) in NIH/3T3 cells. We observed that both PMA and TGFalpha induced ERK1/ERK2 phosphorylation within 5 min of stimulation. PMA acts upstream of MEK and via activation of protein kinase C (PKC), as GF109203X, a potent PKC inhibitor, and U0126, a MEK inhibitor, abolished its actions on ERK1/ERK2 phosphorylation. TGFalpha did not act via PKC because GF109203X failed to curtail the degree of ERK1/ERK2 phosphorylation in these cells. DHA alone failed to induce the phosphorylation of these mitogen-activated protein (MAP) kinases; however, this fatty acid significantly curtailed the PMA- but not TGFalpha-induced MAP kinase enzyme activity and phosphorylation in NIH/3T3 cells. Furthermore, we observed that DHA significantly inhibited PMA-induced translocation of two PKC isoforms, PKC alpha and PKC epsilon, from cytosol to plasma membrane. Interestingly, DHA failed to inhibit the PMA-induced translocation PKC delta isoform in these cells. Furthermore, DHA decreased PMA-induced proliferation of NIH/3T3 cells. In this study, we show for the first time that DHA inhibits MAP kinase ERK1/ERK2) activation and proliferation of NIH/3T3 cells via its inhibitory action on PKC alpha and epsilon isoforms.