Oxidative stress in biological membranes can regulate various aspects of apoptosis, including phosphatidylserine (PS) externalization. It is not known, however, if the targets for these effects are lipids or proteins. Nitric oxide (NO), a bifunctional modulator of apoptosis, has both antioxidant and prooxidant potential. We report here that the NO donor PAPANONOate completely protected all phospholipids, including PS, from oxidation in HL-60 cells treated with 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN), presumably via the ability of NO to react with lipid-derived peroxyl radicals and terminate the propagation of lipid peroxidation. PAPANONOate, however, had no effect on PS externalization or other markers of apoptosis following AMVN. Therefore, PS oxidation is not required for PS externalization during AMVN-induced apoptosis. PS externalization was accompanied by inhibition of aminophospholipid translocase (APT). NO potentiated AMVN inhibition of APT. Treatment with PAPANONOate alone produced modest (20%) inhibition of APT without PS externalization. NO did not reverse AMVN-induced oxidation of glutathione and protein thiols. We speculate that APT was sensitive to AMVN and/or NO via modification of protein thiols critical for functional activity. Therefore, the lipoprotective effects of NO were insufficient to prevent PS externalization and apoptosis following oxidative stress. Other targets such as protein thiols may be important redox-sensitive regulators of apoptosis initiation and execution. Thus, in the absence of significant peroxynitrite formation, NO's antioxidant effects are restricted to protection of lipids, while modification of protein substrates continues to occur.