The poor prognosis of patients with malignant gliomas necessitates the development of novel therapies. Virotherapy, using genetically engineered adenovectors that selectively replicate in and kill neoplastic cells, represents one such strategy. In this study, we examined several oncolytic vectors with modified transcriptional and transductional control of viral replication. First, we incorporated the survivin promoter (S) to drive E1A gene expression. We then modified the adenovirus serotype 5 (Ad5) fiber protein via genetic knob switching or incorporation of peptide ligands to target the following glioma-associated receptors: the Ad3 attachment protein, or CD46, alpha(v) beta(3)/alpha(v)beta(5) integrins, or heparan sulfate proteoglycans. The three conditionally replicative adenoviruses, CRAd-S-5/3, CRAd-S-RGD, and CRAd-S-pk7, were then examined in vitro with respect to transduction efficiency and tissue specificity. The most promising virus was then tested in vivo for evidence of tumor growth inhibition. CRAd-S-pk7 provided the highest level of viral replication and tumor oncolysis in glioma cell lines. At the same time, we observed minimal viral replication and toxicity in normal human brain. Injection of CRAd-S-pk7 inhibited xenograft tumor growth by more than 300% (p < 0.001). Sixty-seven percent of treated mice with intracranial tumors were long-term survivors (>110 days; p < 0.005). Analysis of tumor tissue indicated increased adenoviral infectivity, decreased mitotic activity, and enhanced tumor apoptosis. These findings demonstrate the effectiveness of CRAd-S-pk7 and provide the rationale for further development of this novel oncolytic virus for glioma gene therapy.