Skeletal muscle maintenance, repair and growth are dependent on satellite cell activation. These quiescent cells are activated to proliferate to allow ultimately formation of new myotubes, when for example muscle injury occurs. . Murine C2C12 myoblasts can be induced to differentiate into myotubes and provide a convenient model for evaluating the effects of EPA, an omega-3 fatty acid credited with considerable health benefits, on this process. Previous studies have indicated that EPA can inhibit proteolysis of fully differentiated skeletal muscle myotubes (1,2). For this investigation, C2C12 proliferating myoblasts were induced to differentiate by culture in growth medium containing 2% horse serum. EPA (0.01mM to 0.6mM), complexed with bovine serum albumin, was added to the differentiation medium at the start and myotube formation allowed to progress for 5 days. Indirect immunofluorescence microscopy with anti-myosin primary antibody (MF-20, DSHB University of Iowa) and Alexa-Fluor® 546 goat anti-mouse IgG secondary antibody (Invitrogen), followed by DAPI nuclear counterstaining were performed to evaluate myotube formation. A commercial cytotoxicity assay (CellTiter Blue Assay, Promega) was carried out to quantify cell death. The cytotoxicity results, obtained after 5 days differentiation, show two distinct dose-dependent effects of EPA on cell death. At EPA concentrations >0.4mM, cell death increases significantly over that of untreated cells (P < 0.05). In contrast, at EPA doses between 0.01mM and 0.3mM, cell death is significantly reduced compared to untreated cells (P < 0.05). During myotube differentiation in the presence of low concentrations of EPA It was observed that there was an approximately 50% reduction in cell death. Data from immunofluorescence studies of cell death confirmed these findings. In conclusion, our data suggest that low doses of EPA exert a protective effect against cell death during myotube formation. Possible mechanisms could include an effect of EPA on the PI3K/AKT signalling pathway as this pathway has previously been shown to be involved with mediation of differentiation (4). Although the nature of this effect is currently being investigated, a previous study has demonstrated that EPA, administered at a similar dosage, can inhibit apoptosis of fully differentiated myotubes (3). Our findings support a potential therapeutic application for EPA during skeletal muscle regeneration following injury.