Background: Oncolytic adenoviruses are under study as a potential anticancer treatment. Their preclinical antitumor activity; however, has not been effectively translated to the clinic. A contributing factor may be the clinical relevance of immunodeficient mouse tumor xenograft models used to study these vectors. While these demonstrate viral replication in the xenograft, they are limited by the lack of natural host immune responses to the virus, associated toxicity, dissemination and systemic viral replication. Cotton rats (Sigmodon hispidus) are semi-permissive for the replication of human adenovirus and have been used to study these vectors. Until recently this model lacked a transplantable tumor to assess oncolytic activity. We characterized three syngeneic transplantable cotton rat tumor cells lines that support adenovirus replication and oncolysis in this immunocompetent rodent model. Methods: Three tumor cell lines: CCRT, LCRT and VCRT were derived from a spontaneously arising osteosarcoma, and fibrosarcomas of the breast and jaw in animals from an inbred cotton rat colony. The ability of the cells to be infected with adenovirus was examined using an adenovirus expressing green fluorescent protein (Ad.GFP). Viral burst assays were used to determine titers of infective adenovirus produced by each cell line at 4 and 72 hours following infection with wild type adenovirus type-5 (Ad5). Transmission electron microscopy (TEM) was used to visualize the relative number of virions at each time point. We also evaluated replication and oncolysis of the cell lines in vivo. Results: Using Ad.GFP, the cell lines were readily infected and demonstrated GFP expression comparable to human A549 cells. All cell lines showed significant higher (P<0.05) intracellular titers of adenovirus and in supernatants 72 hours after infection compared to 4 hours after infection with Ad5 indicating viral replication and increased number of functional virions. In addition, all the cell lines exhibited greater expression of adenoviral hexon protein at 72 hours than at 4 hours after infection, indicating late transcriptional unit gene activation. The cotton rat cell lines formed tumors in cotton rats when injected subcutaneously. On days 5 and 8, PCR demonstrated 2 to 3-log greater number of Ad5 genome copies in tumors injected with Ad5 compared to those injected with a non- replicating adenovirus (Ad.null). This was consistent for CCRT, LCRT and VCRT. Ad5 slowed tumor growth in cotton rats subcutaneously injected with CCRT and VCRT cells. Despite in vitro oncolysis, no anti-tumor effect was seen in the LCRT tumors with the virus rapidly cleared from the tumor. Conclusions: CCRT, LCRT and VCRT, three novel transplantable cotton rat tumor-derived cell lines demonstrate replication of Ad5 both in vitro and in vivo. In addition, CCRT and VCRT demonstrated in vivo oncolysis. The varied antitumor effect in this model mirrors the results of human clinical trials highlighting the potential relevance of these cotton rat tumor models for assessing replication competent adenovirus vectors.