Regulatory T cells (Treg) are a distinct group of T lymphocytes with immunosuppressive properties that serve normally to prevent harmful autoimmune responses. However, Tregs can also interfere with beneficial immune responses such as anti-tumor and anti-viral immunity in humans and rodents. Given the overall importance of Tregs, it is likely that they play an important role in diseases of dogs as well. However, at present reagents required for identification of Tregs in dogs are not available. Therefore, we investigated whether expression of FoxP3, a transcription factor that is highly expressed in Tregs in humans and rodents could also be used to identify Tregs in dogs. We found that a cross-reactive FoxP3 antibody identified a subset of CD4+ T cells in blood and lymph nodes of dogs. By flow cytometry the mean percentage of FoxP3+CD4+ T cells in normal dogs was 4.3% in blood and 9.8% in the lymph nodes. In dogs with cancer, there was a significant increase in numbers of Treg in blood (7.5%) and tumor-draining lymph nodes (17.1%) compared to age-matched healthy control dogs. We also found that FoxP3+CD4+ T cells in dogs could be significantly expanded in vitro by TCR activation together with addition of TGF-β and IL-2. Treated cells also significantly increased expression of TGF-β and IL-10mRNA. We conclude from these studies that a cross-reactive FoxP3 antibody can be used to identify Tregs in dogs and that this reagent may serve as a useful tool for investigating the role of Treg in a variety of diseases of dogs.