YKL-1, E1B 55 kDa-deleted recombinant adenovirus vector, capable of harboring a transgene casette of up to 4.9 kb, was newly constructed by reintroducing E1A and E1B 19 kDa into E1/E3-deleted adenoviral vector with a homologous recombination in E. coli. Virus replication and cytotoxicity were dramatically attenuated in all 3 different types of normal human cells. In contrast, YKL-1 efficiently replicated and induced cytotoxicity in most cancer cells, especially Hep3B and C33A cells with an inactivating p53 mutation. However, both H460 and HepG2 exhibited intermediate sensitivity to YKL-1, which was between that of Hep3B or C33A and normal human cells. The YKL-1 and DNA damaging agent, camptothecin effectively induced p53 in H460 and HepG2 as well as in normal cells. Furthermore, YKL-1 effectively prohibited both Hep3B and C33A tumor growth in nu/nu mice in a dose-dependent manner. H/E staining and TUNEL assay indicated a largely distributed necrotic area and apoptosis on its periphery. This study, therefore, indicates that YKL-1, possesses promising potential as an oncolytic adenoviral vector, which acts partially in a p53-dependent manner.