A recombinant adenovirus expressing Escherichia coli cytosine deaminase (AdCD) was constructed with the purpose of exploring its utility for the treatment of breast cancer. Infection of the human breast cancer cell line, MDA-MB-231, with AdCD resulted in high levels of cytosine deaminase enzyme activity. MDA-MB-231 cells infected with AdCD were 1000-fold more sensitive to 5-fluorocytosine (5-FC) than cells infected with a control adenovirus. Cell mixing experiments indicated that only 10% of AdCD-infected cells in a population were needed to induce complete cytotoxicity of noninfectious cells exposed to 5-FC. This suggests that bystander effects play an important role in AdCD-mediated cytotoxicities. Direct injection of AdCD into human breast MDA-MB-231-derived tumors grown as xenografts in nude mice, followed by daily intraperitoneal injection 5-FC was sufficient to inhibit tumor growth. These results suggest that in vivo gene therapy for breast cancer using AdCD is feasible.