Top of pageAbstract Adenoviral vectors offer promise for gene therapy of various diseases. One recent development in this area is conditionally replicative adenoviruses (CRAds) which selectively kill cancer cells through tumor-specific replication. To maintain safety and practically apply these agents in a clinical context, a non-invasive detection system of viral replication is required. Current adenovirus detection methods used in virotherapy clinical trials include in situ hybridization, immuno-histochemistry, electron microscopy, and DNA amplification. Although these techniques are useful for examining the presence of adenovirus in a laboratory setting, they are not suitable for monitoring dynamic events and isolated such as viral replication in clinical context. These methods can only provide static data on replication since they rely on invasive biopsies. To address these issues, we hypothesized that expression of a reporter gene from a replicative adenovirus would yield valuable information on viral replication and localization. We chose a strategy to incorporate the EGFP fluorescent protein into the E3 region so that transgene expression under the adenovirus major late promoter would closely mimic the viral replication profile. In addition, the chosen fluorescent reporter protein has potential to be detected noninvasively. We constructed Ad5ΔE3ADPF by deleting proteins 6.7K, gp19K, RIDα (10.4K), RIDβ (14.5K), and a portion of 12.5K from the E3 region while retaining the adenovirus death protein (ADP) since this viral product is required for efficient cell lysis and viral release. This construct has been reported to overexpress ADP and cause enhanced cytopathic effect. Using this construct, we generated six different vectors with EGFP in six different configurations (forward / reverse directions and three reading frames). Vectors with the EGFP cassette in the forward direction (F0, F1, F2: left to right) expressed EGFP 12 hours after infection in A549 (adenovirus permissive human lung carcinoma cell line). On the other hand, no EGFP expression was seen in BNL1NGA.4 (mouse hepatoma cell line) in which human adenovirus can infect but not replicate productively. Of the three frames, F1 showed the clearest correlation of EGFP expression with viral replication. Vectors with reverse direction cassettes (R0, R1, R2: right to left) did not demonstrate any EGFP expression. In the context of cytocidal effect, viruses with ΔE3ADP structure showed same level of cell killing effect as Ad5 wild type in A549 cells. Our data suggest that our novel imaging system may provide a simple and convenient way to monitor viral replication in preclinical and clinical settings. Additionally, deletion of the E3 region while remaining ADP would yield more cloning capacity for transgenes in a replicative adenovirus without hampering cell killing effect. We are currently testing the functionality of our system in several CRAd configurations for noninvasive imaging purposes.