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An E1B-defective adenovirus named r1/Ad was constructed by homologous recombination. The construction, selection and propagation of recombinant virus was done in the human embryonic kidney 293 cells (HEK293). The in vitro study demonstrated that the recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as the human glioblastoma tumor cells (U251) and human bladder tumer cells (EJ) but not in the normal cells with functional p53 such as the human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated that the U251 cells were more sensitive to the infection of r1/Ad than that of EJ cells under identical conditions. In this paper, it was found that r1/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus. This may help to determine the safety of using any E1B-defective adenoviruses in cancer gene therapy.