The 55-kilodalton (kDa) protein from the E1B-region of adenovirus binds to and inactivates the p53 gene, which is mutated in half of human cancers. We have previously shown that the replication and cytopathogenicity of an E1B, 55-kDa gene-attenuated adenovirus, ONYX-015, is blocked by functional p53 in RKO and U2OS carcinoma lines. We now report that normal human cells were highly resistant to ONYX-015-mediated, replication-dependent cytolysis. In contrast, a wide range of human tumor cells, including numerous carcinoma lines with either mutant or normal p53 gene sequences (exons 5–9), were efficiently destroyed. Antitumoral efficacy was documented following intratumoral or intravenous administration of ONYX-015 to nude mousehuman tumor xenografts; efficacy with ONYX-015 plus chemotherapy (cisplatin, 5-fluorouracil) was significantly greater than with either agent alone.