Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.