CD8 is a heterodimeric cell surface glycoprotein expressed primarily on thymocytes and a subpopulation of mature T lymphocytes. It binds to the invariant part of the major histocompatibility complex class I molecule and participates in antigen recognition by the major histocompatibility complex class I restricted T cells. As in mammalian species, the majority of chicken thymocytes express both CD4 and CD8, whereas peripheral T cells are either CD4- or CD8-positive. We have created a panel of mouse monoclonal antibodies detecting different cell surface epitopes on chicken CD8. The antibodies precipitate a 32-34 kDa dimeric protein from surface labelled thymocytes under reducing conditions. The identical N-deglycosylation pattern confirms that these MoAb precipitate the same heterodimeric molecule from chicken thymocyte lysates. Binding of 11-38 and 11-39 MoAb to peripheral blood T cells is totally inhibited by 11-39 and previously characterized CT8 and EP72 MoAb, further confirming their CD8 specificity. CD8 alpha-chain specificity of MoAb 11-39, 11-38, 11-30 and 11-13 is conclusively proven by staining COS-cells transfected with a plasmid containing CD8 alpha cDNA. However, MoAb 11-13, 11-30 and 11-38 do not compete with MoAb 11-39 in binding to CD8. These results demonstrate recognition of different epitopes by these MoAb. Monoclonal antibodies detecting novel epitopes on chicken CD8 provide a valuable tool for further studies on T cell development.