Effective anti cancer strategies necessitate the use of agents that target tumor cells rather than normal tissues. In this study, we constructed a tumor-selective adenovirus, Delta24, that carries a 24-bp deletion in the E1A region responsible for binding Rb protein. Immunoprecipitation analyses verified that this deletion rendered Delta24 unable to bind the Rb protein. However, titration experiments in 293 cells demonstrated that the Delta24 adenovirus could replicate in and lyse cancer cells with great efficiency. Lysis of most human glioma cells was observed within 10 - 14 days after infection with Delta24 at 10 PFU/cell. In vivo, a single dose of the Delta24 virus induced a 66.3% inhibition (P<0.005) and multiple injections, an 83.8% inhibition (P<0.01) of tumor growth in nude mice. However, normal fibroblasts or cancer cells with restored Rb activity were resistant to the Delta24 adenovirus. These results suggest that the E1A-mutant Delta24 adenovirus may be clinically and therapeutically useful against gliomas and possibly other cancers with disrupted Rb pathway.