To identify chicken IL-2R alpha chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL-2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.