Purpose: To determine the maximum tolerated dose (MTD), toxicity spectrum, clinical activity, and biological effects of the tropism-modified, infectivity-enhanced conditionally replicative adenovirus (CRAd), Ad5-Δ24-Arg-Gly-Asp (RGD), in patients with malignant gynecologic diseases. Experimental Design: Cohorts of eligible patients were treated daily for 3 days through an i.p. catheter. Vector doses ranged from 1 × 10 9 to 1 × 1012 viral particles per day. Toxicity was evaluated using CTCv3.0. CA-125 and Response Evaluation Criteria in Solid Tumors (RECIST) criteria were used to determine clinical efficacy. Corollary biological studies included assessment of CRAd replication, wild-type virus generation, viral shedding, and neutralizing antibody response. Results: Twenty-one patients were treated. Adverse clinical effects were limited to grade 1/2 fever, fatigue, or abdominal pain. No vector-related grade 3/4 toxicities were noted. No clinically significant laboratory abnormalities were noted. The maximum tolerated dose was not reached. Over a 1 month follow-up, 15 (71%) patients had stable disease and six (29%) had progressive disease. No partial or complete responses were noted. Seven patients had a decrease in CA-125; four had a >20% drop. RGD-specific PCR showed the presence of study vector in ascites of 16 patients. Seven revealed an increase in virus after day 3, suggesting replication of Ad5-Δ24-RGD. Minimal wild-type virus generation was detected. Viral shedding studies showed insignificant shedding in the serum, saliva, and urine. Antiadenoviral neutralizing antibody effects were prevalent. Conclusions: This study, the first to evaluate an infectivity-enhanced CRAd in human cancer, shows the feasibility, safety, potential antitumor response, and biological activity of this approach in ovarian cancer. Further evaluation of infectivity enhanced virotherapy approaches for malignant gynecologic diseases is warranted. ©2010 AACR.