Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.