To gain further insight into the mechanism of action of the immunosuppressant cyclosporin A (CyA), we investigated the influence of CyA on proliferative responses of human T lymphocytes, induced via different membrane molecules. As was previously shown, activation of T cells via the T cell receptor (Ti)/CD3 complex with an anti-CD3 monoclonal antibody was inhibited by CyA. Likewise, triggering of T lymphocytes via the alternative, CD2(T11)-mediated pathway of activation was strongly inhibited. In contrast, responses induced by phorbol myristate 13-acetate (PMA; 100 ng/ml) or the combination of an anti-CD28 monoclonal antibody and a suboptimal concentration of PMA (1 ng/ml) were found to be insensitive to CyA. CyA-induced inhibition of both anti-CD3- and anti-CD2-mediated proliferation could not be reversed by addition of either PMA (1 ng/ml) or anti-CD28. An increase in the intracellular free Ca2+ concentration [( Ca2+]i) is an early event observed after stimulation of T cells via CD3 or CD2, whereas stimulation with PMA and anti-CD28 does not lead to a rise in [Ca2+]i. This suggests that the inhibitory action of CyA is related to Ca2+-dependent signaling pathways. Since we observed that CyA does not interfere with anti-CD3- or anti-CD2-induced increases of [Ca2+]i, our data suggest that CyA-mediated inhibition is related to a later event in these intracellular signaling pathways.