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 15 T8+clones were highly cytotoxic for the sensitizing alloantigen. In contrast, only two of the 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 α -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 that cytotoxic T4+subpopulations might be important in human transplantation and autoimmune disorders.