Oxidative stress is a potential component of the final common pathway leading to apoptosis following many diverse stimuli. Here, we document that the oxidant paraquat caused apoptosis in mouse 32D cells. We examined early paraquat-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid (cis-PA) into phospholipids and high-performance liquid chromatography separation of specific phospholipid classes. Paraquat induced peroxidation of cis-PA primarily in phosphatidylserine (PS) and to a lesser extent in phosphatidylinositol (PI) within 2 h. The selective oxidation of PS occurred before signs of cytotoxicity and preceded the externalization of PS as assessed by annexin V binding. Overexpression of Bcl-2 afforded significant protection against paraquat-induced apoptosis, early PS and PI oxidation, and PS externalization but not the ultimate formation of high-molecular-weight DNA fragments. Therefore, both selective phospholipid peroxidation and DNA damage occurred after paraquat exposure, but only the former was specifically associated with apoptosis. We suggest Bcl-2 may inhibit oxidant-induced apoptosis by preventing the peroxidation of specific membrane phospholipids.