Murine T and B splenocytes were incubated with antibodies that recognize CD3 or surface IgM. These antibodies induced proliferation of their respective target cells. Once stimulated via their receptors, the proliferation of both CD4+ and CD8+ T but not B lymphocytes was inhibited by class I-specific antibodies or their monovalent Fab' fragments. The inhibition of proliferation was dependent on the site on class I molecules recognized by the antibodies used, with the alpha 1/alpha 2 domains of H-2K molecules representing the major site for inhibition. Only soluble antibody-mediated proliferation could be inhibited by class I-directed antibodies; proliferation induced by CD3-specific antibody immobilized on plastic was not inhibited. Primary allogeneic MLR was also inhibited by class I-specific antibodies. In contrast, neither secondary allogeneic MLR, secondary Ag-specific responses, nor proliferation of CTL clones or tumor cell lines were inhibited by class I-specific antibodies. These results suggest a role for class I molecules in regulation of TCR/CD3- but not surface IgM-mediated cell signaling, which depends on the form of stimulation and the stage of differentiation of T cells.