We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fl uorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fl uorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verifi ed by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confi rmed by in vitro gamma camera and fl uorescent microscopy imaging. Comparison of tk-mRFP virus to singlemodality controls revealed similar replication effi ciency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fl uorescence imaging demonstrated a strong correlation between the intensity of fl uorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fl uorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus effi cacy in human trials. © 2009 BC Decker Inc.