Previous work demonstrated that exogenous expression of Rybp (Ring 1 YY1-binding protein or DEDAF) kills tumor but not non-transformed cells. This tumor-preferential killing activity could be exploited in a gene therapy approach to treat cancer. To test the potential of viral-mediated delivery of Rybp as an anticancer treatment, we generated an adenovirus expressing Rybp (Ad-Rybp). Infection with Ad-Rybp inhibits the proliferation of a range of tumor cell lines, but has no effect on normal cell types. This inhibition of proliferation is the result of the induction of apoptosis, consistent with reports that Rybp regulates apoptosis. Combined Ad-Rybp infection and etoposide treatment resulted in an additive cytotoxic effect in the osteosarcoma cell line U20S. Furthermore, Ad-Rybp infection synergistically cooperates with the death receptor ligand, tumor necrosis factor-alpha, in the induction of apoptosis. These results suggest that Ad-Rybp may have clinical applicability, either alone or in combination with other agents for the treatment of cancer.