Human peripheral blood lymphocytes are activated to proliferate and form colonies in semisolid agarose after only brief exposure (24 h) to allogeneic lymphocytes in liquid culture. Colony development is independent of cell contact and exogenously supplied growth factors. The colonies are composed of T cells which form E rosettes with sheep erythrocytes and do not express surface immunoglobulin. Pooled colonies derived from 24-h allogeneic stimulation of HLA-D homozygous typing cells (HTC) express the appropriate HLA-D-associated DR antigens, as measured by complement-mediated cytotoxicity and by the reactivity of anti-DRw alloantisera after absorption with HLA-D homozygous B lymphoid cell lines. Some of the antisera give extra reactions with HTC colony cells that have not been previously detected on B lymphoid cell lines and purified peripheral B and T cell populations, suggesting that these sera may also contain antibodies against antigens expressed only on alloactivated T lymphocytes. Single colonies removed from agarose undergo extensive proliferation (20-30 generations) in liquid culture with the aid of T cell growth factor provided by conditioned medium. The majority of these T lymphocyte "clones" tested display specific cytolytic effector function, indicating that within 24 h of exposure to alloantigen, responder lymphocytes that subsequently form colonies in agarose are committed to antigen-specific cytotoxic activity; memory for the priming alloantigens is retained after months of culture with reexposure to antigen. This is the first demonstration that primary alloreactive clones of human T lymphocytes express HLA-DRw antigens and are capable of antigen-specific cytotoxic function.