Revised Manuscript Received February 22, 1994® abstract: Oxidizability of lipids in homogeneous solution varies linearly with theextent of their unsaturation. In vitro cellular, as well as in vivo, studies of oxidizability have generally relied upon chemical indicators of peroxidation such as thiobarbituric acid-reactive substances. To examine the oxidizabilityof lipids in cells, we have measured oxygen uptake and, using electron paramagnetic resonance spin trapping with a-(l-oxo-4-pyridyl)-7V-íerí-butylnitrone (POBN), the real time generation of lipid-derived free radicals. We haveused our experimental in vitro cellular lipid modification model to examine the rate and extent of lipid peroxidation versus the degree of lipid unsaturation in L1210 murine leukemia cells. Lipid peroxidation was stimulated using the prooxidants iron, ascorbate, and the ether lipid compound lO-octadecyl-2-O-methyl-/-ac-glycero-3-phosphocholine. We did a total cellular lipid analysis to determine the number of lipid carbon-carbon double bonds contained in LI210 cells enriched with eight fatty acids of different degrees of unsaturation. We found in cellular lipids that (i) lipid chain length had no apparent effect on the rate or extent of radical formation;(ii) the maximum amount of lipid radical generated increases with the total number of¿¡ s-allylic positions in the cellular lipids; and, most importantly,(iii) the rate of cellular lipid peroxidation increases exponentially with the number of¿ us-allyhc positions. Our quantitative results clearly demonstrate, for the first time, that the number ofbij-allylic positions contained in the cellular lipids of intact cells determines their susceptibility, ie, oxidizability, to free radical-mediated peroxidative events.