A monoclonal antibody (mAb), P4A10, was made to the canine interleukin-2 receptor alpha chain (IL-2Rα; p55; Tac antigen; CD25) to facilitate studies of canine regulatory T-cells (Treg). By non-reduced Western blot, P4A10 bound to a 55 kDa protein, the size of human IL-2Rα. In flow cytometry assays, it reacted with a minor population of circulating dog CD3 +CD4 + T-cells and the majority (>60%) of in vitro PMA-Ionomycin (PMA-IO)-activated canine CD3 + T-cells. P4A10 recognized a hematopoietic cell population enriched for FoxP3+ cells as measured by flow cytometry. The P4A10-selected fractions of T-cells had significantly increased copy numbers of CD25, FoxP3, IL-10, and TGFβ as detected by RT-PCR (reverse transcriptase-PCR) compared to the negative fractions. The P4A10-selected cells inhibited 3H (tritiated) thymidine incorporation in a mixed leukocyte reaction (MLR) containing responders of the same origin. P4A10-selected T-cells from fresh peripheral blood mononuclear cells had less FoxP3 ( p = 0.07) by qRT-PCR (quantitative RT-PCR) and were less suppressive ( p = 0.01) than in vitro alloantigen-activated Treg. The mAb P4A10 is specific for canine CD25 and can be used to facilitate studies of CD25+FoxP3+ Treg in this clinically relevant large animal model.