Chemotherapeutic drug-induced apoptosis of human malignant glioma cells involves the death receptor-independent activation of caspases other than caspases 3 or 8 (Glaser et at, Oncogene 18, 5044-5053, 1999), Here, we report that caspases 1, 2, 3, 7, 8, and 9 are constitutively expressed in most human malignant glioma cell lines. Cytotoxic drug-induced apoptosis involves delayed activation of caspases 2, 7, and 9, but not 8 and 3, and is blocked by a broad spectrum caspase inhibitor, zVAD-fmk, Cytochrome c release from mitochondria precedes caspase activation during drug-induced apoptosis and is unaffected by zVAD-fmk or ectopic expression of the viral caspase inhibitor, crm-A In contrast, ectopic expression of BCL-X-L prevents drug-induced cytochrome c release, caspase activation and cell death. Thus, cancer chemotherapy targets the mitochondrial, caspase-dependent death pathway in human malignant glioma cells.