Alloreactive human T lymphocytes were cloned in soft agar or by limiting dilution and subsequently propagated with interleukin 2 and alloantigen for 8 months or more. By indirect immunofluorescence every clone was reactive with anti-Ia antibodies as well as the T cell-specific antibodies anti-T3 and anti-T11 and expressed either T4 or T8 antigens. All 15T8+ clones were highly cytotoxic for the sensitizing alloantigen. In contrast, only two of seven T4+ clones mediated cytotoxic effector function. The specificity of T4+ and T8+ clones and subclones was analyzed on a panel of typing cells and by antibody blocking studies of major histocompatibility complex (MHC) determinants on the stimulating alloantigen. It was found that T8+ clones killed targets that shared class I MHC antigens (HLA-A,B) with the original stimulator cells whereas cytotoxic T4+ clones were directed at class II MHC antigens (Ia-related). Preincubation of the allogeneic target cell with a monoclonal antibody to a nonpolymorphic HLA alpha-chain determinant inhibited killing by the T8+ clones but did not affect T4+ cytotoxic function. In a reciprocal fashion, anti-IA antibodies to common framework structures on the same target cell blocked killing by T4+ but not by T8+ clones. These results indicate that T4+ and T8+ T lymphocytes have receptors for different classes of MHC antigens and suggest tha cytotoxic T4+ subpopulations might be important in human transplantation and autoimmune disorders.