Replication-competent adenovirus-mediated suicide gene therapy is an investigational cancer treatment in which an oncolytic adenovirus armed with chemo-radiosensitizing genes is used to destroy tumor cells. Previously, we evaluated the toxicity and efficacy of this approach in two clinical trials of prostate cancer using a first-generation adenovirus. Here, we report the toxicity and preliminary efficacy of this approach in combination with intensity-modulated radiotherapy (IMRT) in patients with newly diagnosed prostate cancer using an improved, second-generation adenovirus. The investigational therapy was associated with low toxicity, and there were no dose-limiting toxicities or treatment-related serious adverse events. Relative to a previous trial using a first-generation adenovirus, there was no increase in hematologic, hepatic, gastrointestinal (GI), or genitourinary (GU) toxicities. Post-treatment prostate biopsies yielded provocative preliminary results. When the results of two similar trials were combined, 22% of evaluable patients were positive for adenocarcinoma at their last biopsy, which is better than expected (>or=40%) for this cohort of patients (P=0.038). When the results were categorized by prognostic risk, most of the treatment effect was observed in the intermediate-risk group, with 0 of 12 patients (0%) being positive for cancer at their last biopsy (P<0.01). These results further demonstrate the safety of this investigational approach and raise the possibility that it may have the potential to improve the outcome of conformal radiotherapy in select patient groups.