We have previously found that 22:6n-3 promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic neuronal cell death. We have also demonstrated that n-3 fatty acid deficiency or chronic alcohol exposure markedly decreased the PS content specifically in neuronal cells where 22:6n-3 was highly enriched. During this period, the underlying mechanisms of the effect of polyunsaturates on the survival of neuronal cells was investigated using apoptotic models established by serum starvation or staurosporin treatment. As was the case with serum starvation, apoptotic cell death induced by staurosporin treatment was significantly reduced by the enrichment of Neuro 2A cells with 22:6n-3. Under conditions where PS accumulation is inhibited, 22:6n-3 enrichment was not effective, suggesting that PS plays an important role in the protection of staurosporine-induced apoptosis. The protective effect of 22:6n-3 enrichment was abolished by PI3-kinase inhibitors. Consistently, phosphorylation of Akt at ser-473, which was significantly reduced by staurosporine treatment, was restored by the 22:6n-3 enrichment. These results suggest that PS accumulation promoted by 22:6n-3 plays an important role in PI3-kinase/Akt signaling and deprivation of this fatty acid either due to n-3 fatty acid dietary deficiency or due to chronic alcoholism may have adverse effects on neuronal survival. In an attempt to elucidate the molecular mechanism underlying the effect of 22:6n-3-enriched cell membranes in signal transduction, we initiated a study of protein conformation during this period. Using intra-molecular cross linking and mass spectrometric anaysis, we were able to establish a method to evaluate the changes in three dimensional structure of proteins. This approach will soon be applied to characterize protein conformational changes due to molecular interactions involved in cell signaling processes.