Glioblastoma multiforme (GBM) is a highly malignant brain tumor that is resistant to conventional radiotherapy and chemotherapy. The median survival time of patients with GBM has remained less than 2 years despite concerted efforts to improve therapy. As a new approach to treat GBM we generated retroviral particles encoding mutant survivin for transduction of glioma cells. We demonstrate here that retroviral over-expression of a nonphosphorylatable Thr-34 → Ala mutant of survivin (survivinT34A), in the glioma cell lines U373 and H4 resulted in a marked increase in the percentage of cells bearing multiple nuclei, which was accompanied by significantly decreased cell proliferation, and in greater numbers of cells with hypodiploid DNA content. Administration of the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl- ketone did not reduce the cell death rate. Yet increased nuclear translocation of apoptosis-inducing factor (AIF) was observed in cells transduced with survivinT34A, indicating caspase-independent cell death. Transduction of retroviral vectors encoding wild-type survivin also led to the appearance of multinuclear cells. In contrast to mutant survivin, overexpressed wild-type survivin did not increase the cell death rate and no enhanced nuclear AIF translocation was observed. We suggest that retroviral vectors delivering mutant survivinT34A might be employed for the treatment of glioblastoma.