Oxidative stress has been implicated as a causal factor in a wide variety of neurodegenerative diseases. To investigate the direct consequences of oxidative damage on myelin-forming cells, we have exposed oligodendrocytes to hydrogen peroxide. Cytotoxicity was assessed in glial cultures by neutral red (NR) and MTT assay, and half-maximal cytotoxicity was reached after a 30-min application with 100-200 microM H2O2 during a 16-24-h recovery period. The cytotoxic effect could be partly abolished by the simultaneous incubation with N-acetyl-l-cysteine, an antioxidant and precursor of glutathione. In purified mature oligodendroglia cultures (7 div), metabolic activity as determined by the MTT assay, was impaired directly after the treatment with H2O2, and only slightly further enhanced during the 24-h recovery period. Morphological inspection revealed that oligodendrocytes in either the presence or the absence of astrocytes were specifically susceptible to free radical damage, the membranous sheets were disrupted, membranous blebs appeared, and fragmented nuclei were seen. Similar changes were induced by treatment with menadione or staurosporine. The data show that brief exposure to H2O2 induced cell death via apoptosis. This death occurred over a period of 24 h and was accompanied by the appearance of fragmented and condensed DAPI-stained nuclei and internucleosomal DNA cleavage. Concomitantly, as investigated by RT-PCR analysis, the transcriptional activity of c-fos and c-jun was stimulated, without altering mRNA expression of the myelin-specific genes MBP, MAG, and PLP. Thus, oxidative stress in oligodendrocytes leads to the onset of programmed cell death, involving the transcriptional activation of the immediate-early genes c-fos and c-jun.