Introduction: The pleiotropic cytokine tumour necrosis factor-alpha (TNF-α) and the cytosolic phospholipase A₂ (cPLA₂) are involved in the cellular inflammatory response and ischaemic injury. During ischaemia TNF-α is endogenously generated by muscle (1,2) activating cPLA₂ (3). In addition to this, low dose TNF-α has been shown to confer cytoprotection in muscle(2). Furthermore, cPLA₂ activation is known to result in the generation of mediators of cellular injury, apoptosis and necrosis(4). We have therefore investigated the role of cPLA₂ in low dose TNF-α mediated cytoprotection in a muscle cell line. Methods: Murine C2C12 myoblasts were grown to 50% confluency and differentiated into myotubes. Myotubes were then included in one of the following experimental groups: normoxic control; ischaemic control; ischaemic preconditioning (IPC); TNF-α preconditioning (TNF-PC); and TNF-PC with AACOCF3 (cPLA₂ inhibitor, TNF-I). Following a simulated ischaemic insult cell viability was determined using trypan blue exclusion. In addition cPLA₂ phosphorylation state and cellular localisation was assessed using western blotting and fluorescence immuno-histochemistry combined with 3-D imaging techniques and analysis. Results: Exposure to simulated ischaemia reduced cell viability when compared to the normoxic control (from 81.6±2.0% to 24.8±3.9%, n≥3, p<0.05). IPC, TNF-PC and TNF-I prevented the ischaemia induced decrease in cell viability (57.6±3.7%, 65.8±3.4% and 53.7±1.1%, n≥3, p<0.05 vs. ischaemic control). cPLA₂ phosphorylation was reduced in the ischaemic control and TNF-PC groups (41.1±0.5% and 45.3±5.0% respectively, n≥3, p<0.05 vs. normoxic control). Phosphorylation was further reduced in the IPC group (20.3±5.2%, n≥3, p<0.05). In addition to these changes, differences were seen in the cellular localisation of the cPLA₂ in the various groups, with a predominantly cytosolic distribution seen in the normoxic control and ischaemic control groups, and a more nuclear localisation seen in the IPC and TNF-PC groups. Conclusion: This data suggests an additional level of regulation in the TNF – cPLA₂ axis, with a possible role for cPLA₂ in deciding cell fate.