The functional effects resulting from CD4 and CD8 perturbation were analyzed by using a CD4+CD8+ clone and anti-CD4 and anti-CD8 monoclonal antibodies. Perturbation of CD8, but not CD4, by soluble antibody resulted in the inhibition of CD3-T cell receptor (CD3-Ti) triggering as determined by flow cytometric measurements of intracellular free Ca2+ concentrations. In addition, the CD3-T cell receptor-mediated cytotoxic function of the CD4+CD8+ clone was inhibited by anti-CD8, but not by anti-CD4. These results suggest that CD8, but not CD4, was functionally associated with CD3-Ti on the CD4+CD8+ clone. Although CD4 perturbation did not affect CD3-Ti-mediated activities, it resulted in the inhibition of the interleukin 2-dependent proliferation of this clone. Perturbation of CD8 did not affect the interleukin 2 dependent proliferation of the CD4+CD8+ clone. On the other hand, CD4 molecules of another CD4+CD8- clone unlike those of the CD4+CD8+ clone, were clearly linked to T cell receptor function. These results indicate that CD4 perturbation can result in two distinct regulatory activities; one involves the regulation of CD3-T cell receptor function, whereas the other is not directly associated with CD3-T cell antigen receptor function. The data are also consistent with the notion that CD4 and CD8 do not merely function as recognition and adhesion elements for accessory cell major histocompatibility complex molecules, but have a direct role in the regulation of T cell activation.