Conditionally replicative adenoviruses (CRAds) are engineered to replicate only in the target tissue and destroy tumor through their cytopathic effect. Because of restricted in vivo replication, it is difficult to model behavior of human Ad5-based vectors in animal subjects. To circumvent this, we developed a "syngeneic" canine CRAd based on canine adenovirus type 2 (CAV2) transcriptionally targeted to canine osteosarcoma (OS) cells. Canine OS is an outstanding model of human OS and is the most common primary bone tumor of dogs. Because conventional therapies extend median survival by approximately 6-8 months, canine OS remains a serious therapeutic challenge shared by human OS patients. Prior to using any CRAd for clinical trials in dogs, we sought to examine the effects and safety of administration of OS-targeted CAV2 CRAd in normal dogs. Short-term physiologic indicators of stress and shock, as well as gross and histological changes in a variety of tissues, were examined, and no major signs of virus-associated toxicity were noted. In addition, short-term immunosuppression did not increase CRAd toxicity. This study marks the first administration of a CRAd in an outbred large animal model and is an important milestone in the application of this modality in human patients. © Mary Ann Liebert, Inc.