New mouse monoclonal antibodies which detect CD80 and CD83 were developed to characterize chicken dendritic cells (DCs). The characteristics of these molecules have been studied in human, swine, ovine, feline, and canine but not in chickens. MAbs to chCD80 showed staining of chicken macrophage cell line (HD11), bursa, and spleen. MAbs against chCD80 reduced chCD80’s ability inducing T cell proliferation and IFN-γ production in blast chicken spleen cells, and NO secretion in the HD11. Two mAbs, CD83-159 and CD83-22, reacted with a HD11 and recognized a single 53 kDa protein on Western blots of lysates from LPS-stimulated spleen mononuclear cells. Immunostaining of chicken secondary lymphoid organs identified CD80+ and CD83+cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen monocytes with concanavalin A (Con A) decreased the percentage of CD83+ cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII+ cells compared with cells treated with plus an isotype-matched negative control mAb. These mAbs will be useful for future investigations of DC maturation and mechanisms of action in chickens.