We have studied the in vitro activation of chicken y6 T cells. Both splenic aP and y6 T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4+ cells or ap T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of y6 T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4-or aP TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the y 6 T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-y6 TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-y6 TcR monoclonal antibody, a strong proliferation of y6 T cells was observed. In contrast, both Vgl-and VPZexpressing ap T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either VPl+ or VP2+ T cell subset alone had no negative effect on the proliferation or blast formation of y6 T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4+ aP T cells (both VPl-and VP2-expressing) play a role in the activation and response of chicken y6 T cells.