The effect of polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (22:6n-3; DHA) and arachidonic acid (20:4n-6; AA), on apoptotic cell death was evaluated based on DNA fragmentation and caspase-3 activity induced by serum starvation using Neuro-2A and PC-12 cells. The presence of 20:4n-6 in the medium during serum starvation decreased DNA fragmentation and this initial protective effect was diminished with prolonged serum starvation. The observed protective effect of 20:4n-6 was not affected by the inhibitors of cyclooxygenase (COX) and lipoxygenase. Conversely, 22:6n-3 became protective only after the enrichment of cells with this fatty acid at least for 24 h prior to the serum deprivation. DNA fragmentation as well as caspase-3 activity was reduced in 22:6n-3 enriched cells with a concomitant decrease in protein and mRNA levels. During the enrichment period, 22:6n-3 steadily increased its incorporation into PS leading to a significant increase in the total PS content; the protective effect of 22:6n-3 paralleled the PS accumulation. Neither direct exposure of cells to nor enrichment with 18:1n-9 had any protective effect. In conclusion, it is proposed that 20:4n-6 prevents neuronal apoptosis primarily due to the action of nonesterified 20:4n-6 but 22:6n-3, at least in part, through PS accumulation.