There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4+ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4+ cells were induced to proliferate and to synthesize interleukin 2 (IL 2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL 2-producing clones bearing either CD4 or CD8 on their surface. IL 2 production was induced by mAb B66 in CD4+, but not CD8+, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4+ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4+ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.