We previously established that n−3 FA status in membrane phospholipids influences the biosynthesis and accumulation of PS in neuronal tissues. We also demonstrated that neuronal apoptosis under adverse conditions is prevented by DHA enrichment in a PS-dependent manner. In this study, we examined the effect of a structural analog of DHA, docosapentaenoic acid (22∶5n−6, DPA), which accumulates in neuronal membranes during n−3 FA deficiency. We observed that enrichment of neuronal cells with DPA increased the total PS content in comparison to nonenriched control. However, the increase was significantly less than that observed in DHA-enriched cells, primarily due to the fact that the 18∶0,22∶5n−6 species was not accumulated as effectively as 18∶0,22∶6n−3 in PS. As was the case with DHA, DPA enrichment also protected against cell death induced by staurosporine treatment in Neuro 2A cells, but to a lesser extent. These data indicate that provision of DPA in place of DHA is sufficient neither for fully supporting PS accumulation nor for cell survival. The in vitro interaction between Raf-1 and membrane was affected not only by the PS content but also by the fatty acyl composition in PS. The reduction of PS concentration as well as the substitution of 18∶0,22∶6 with 16∶0,18∶1 in the liposome considerably reduced the interaction with Raf-1. These data suggest that depletion of DHA from neuronal tissues may have a compounding effect on Raf-1 translocation in growth factor signaling. The fact that DPA cannot fully support the protective role played by DHA may provide a basis for the adverse effect of n−3 FA deficiency on neuronal development and function.