Jurkat T cells undergo rapid apoptosis upon stimulation of the Fas/APO-1 (CD95) receptor. We examined the role of the mitogen-activated protein kinase (MAPK) cascade as a negative regulator of Fas-mediated apoptosis. To this end, we used both physiologic and artificial activators of MAPK, all of which activate MAPK by distinct routes. MAPK activity could be efficiently elevated by two T cell mitogens, the lectin PHA and an agonistic Ab to the T cell receptor complex as well as by the type 1 and 2A phosphatase inhibitor, calyculin A, and the protein kinase C-activating phorbol ester, tetradecanoyl phorbol acetate. All these treatments were effective in preventing the characteristic early and late features of Fas-mediated apoptosis, including activation of caspases. Our results indicate that the elevated MAPK activities intervene upstream of caspase activation. The degree of MAPK activation by the different stimuli used in our study corresponds well to their potency to inhibit apoptosis, indicating that MAPK activation serves as an efficient modulator of Fas-mediated apoptosis. The role of MAPK in modulation of Fas-mediated apoptosis was further corroborated by transient transfection with constitutively active MAPK kinase, resulting in complete inhibition of the Fas response, whereas transfection with a dominant negative form of MAPK kinase had no effect. Furthermore, the apoptosis inhibitory effect of the MAPK activators could be abolished by the specific MAPK kinase inhibitor PD 098059. Modulation of Fas responses by MAPK signaling may determine the persistence of an immune response and may explain the insensitivity of recently activated T cells to Fas receptor stimulation.