It has been proposed that an adenovirus with the E1b-55kD gene deleted has a selective advantage in replicating in cancer cells that have mutations in the p53 gene (Bischoff et al., 1996). We have explored this hypothesis in several lung cancer cell lines, and evaluated potential mechanisms that might regulate the replication of Ad338, an E1b-55kD-deleted virus, with the objective of developing a rational approach for targeting gene therapy to lung tumors. Our data show that Ad338 replicates poorly in three lung cancer cell lines with various p53 mutations (H441, H446, and Calu1), yet this virus replicates to a high level in a lung cancer cell line with wild-type p53 (A549) and in a normal lung fibroblast line (IMR90). Viral DNA replication, expression of viral proteins, and shutoff of host cell proteins were not important variables in limiting the replication of the E1b-55kD-deleted virus. However, the cell lines resistant to host cell protein shutoff were also the most resistant to the cytopathic effect induced by mutant and wild-type virus and the only cells to survive for 8 days following infection. The E1b-55kD protein clearly has an important role in viral replication beyond its interaction with p53. Thus, an E1b-55kD-deleted virus cannot be used to specifically target viral replication to p53-mutated lung cancer cells.