Objective.: Oncolytic adenoviruses capable of replication selectively in tumor cells are an appealing approach for the treatment of neoplastic diseases refractory to conventional therapies. The aim of this study was to evaluate the effect of dose and scheduling of a tropism-modified, adenovirus serotype 3 receptor-targeted, Rb/p16 pathway-selective replication-competent adenovirus, Ad5/3-Δ24, against human ovarian adenocarcinoma. As oncolytic viruses and chemotherapy can have synergistic interactions, the antitumor efficacy of Ad5/3-Δ24 was also studied in combination with epirubicin and gemcitabine, common second-line treatment options for platinum-resistant ovarian cancer. Methods.: Orthotopic murine models of peritoneally disseminated ovarian cancer were utilized to compare survival of mice treated with either a single viral dose or weekly delivery. The lowest effective dose of intraperitoneal Ad5/3-Δ24 was determined. Combinations of Ad5/3-Δ24 and gemcitabine or epirubicin were studied in vitro as well as in vivo. Results.: Treatment outcome after administration of a single dose of Ad5/3-Δ24 was as effective as delivery of several weekly doses. Our results also demonstrate that a single intraperitoneal injection of 100 viral particles significantly increased the survival of mice compared to untreated animals. Further, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Conclusion.: These preclinical data suggest that Ad5/3-Δ24 represents a promising treatment strategy for advanced ovarian cancer as a single agent or in combination with chemotherapy. © 2007 Elsevier Inc. All rights reserved.