We have expressed the human CD4 and CD2 molecules in a murine hybridoma, 155.16, that responds to stimulation with human HLA-DR antigens by producing interleukin 2 (IL-2). When stimulated by HLA-DR expressing human cells, the CD4+ and CD2+ hybridomas produce significantly more IL-2 than the parent hybridomas, indicating that CD4 and CD2 are functional. We have used this model system to analyze the role of the CD4 and CD2 surface receptors in T cell activation. Different monoclonal antibodies (mAbs) against CD4 and CD2 were used to define regions of these molecules that may be important for ligand binding and function. Using anti-CD4 mAbs in combination with anti-CD3 mAb, and measuring IL-2 production and mobilization of cellular calcium [( Ca2+]i), we have identified two regions of the CD4 molecule that appear to be important for CD4-dependent functions. Anti-CD4 mAb Leu3a bound in the first immunoglobulin-like domain and appeared to enhance CD4-dependent functions. mAb OKT4F, which bound in or near the second immunoglobulin-like domain, inhibited CD4-dependent functions. The role of CD2 in T cell activation was analyzed by characterizing hybridomas that expressed wild type and mutated CD2 molecules. This analysis has shown that two regions of the external domain of CD2 are involved in binding of lymphocyte-function associated antigen-3 (LFA-3). Furthermore, binding of CD2 to LFA-3 enhanced T cell activation, and this enhancement was dependent on the cytoplasmic domain of the CD2 molecule. These results suggest that the CD2/LFA-3 interaction provides a stimulatory signal for T cell activation.