Glutamate and buthionine sulfoximine (BSO) both reduce intracellular glutathione (GSH) concentration but by different mechanisms, and thereby induce cell death in C6 rat glioma cells. The effects of lipid peroxidation on chromosomal DNA damage during the GSH depletion-induced cell death were assessed. Polyunsaturated fatty acids (PUFA), such as arachidonic acid (AA), gamma-linolenic acid and linoleic acid enhanced lipid peroxidation, induced a loss of membrane integrity and consequently promoted 1-2 Mbp giant DNA fragmentation under both glutamate- and BSO-induced GSH-depletion. Treated C6 cells had 3'-OH termini in their DNA which were recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) analysis. Antioxidants capable of scavenging reactive oxygen species and lipid radicals and iron or copper scavengers inhibited both lipid peroxidation and 1-2 Mbp giant DNA fragmentation, consequently protecting against cell death under GSH depletion. These results suggest that GSH depletion induces lipid peroxidation and leads to 1-2 Mbp giant DNA fragmentation; and that PUFAs can promote giant DNA fragmentation and 3'-OH termini in chromosomal DNA enhancing lipid peroxidation of C6 cells.