Combining cancer-selective viral replication and simultaneous production of a therapeutic cytokine, with potent "bystander" anti-tumor activity, are hallmarks of the cancer terminator virus (). To expand on these attributes, we designed a next generation that additionally enables simultaneous non-invasive imaging of tumors targeted for eradication. A unique tripartite "theranostic" adenovirus () has now been created that employs three distinct promoters to target virus replication, cytokine production and imaging capabilities uniquely in cancer cells. Conditional replication of the is regulated by a cancer-selective (truncated ) promoter, the therapeutic component, MDA-7/IL-24, is under a ubiquitous () promoter, and finally the imaging capabilities are synchronized through another cancer selective (truncated ) promoter. Using in vitro studies and clinically relevant in vivo models of breast and prostate cancer, we demonstrate that incorporating a reporter gene for imaging does not compromise the exceptional therapeutic efficacy of our previously reported bipartite . This permits targeted treatment of tumors while monitoring tumor regression, with potential to simultaneously detect metastasis due to the cancer-selective activity of reporter gene expression. This "theranostic" virus provides a new genetic tool for distinguishing and treating localized and metastatic cancers.