Ionizing radiation is known to induce death by apoptosis in malignant and nonmalignant human lymphocytes. The mechanism which initiates the induction of apoptosis has not been identified. Here we demonstrate that a radiation-induced up-regulation of CD95/Fas/APO-1 ligand (CD95-L) is involved in the induction of apoptosis in lymphocytes and lymphoma cells. Using antibodies against CD95-L, we detected a rapid and persistent up-regulation of CD95-L in Jurkat cells and blood lymphocytes after irradiation. Blocking of interactions between CD95/Fas/APO-1 and CD95-L with inhibitory antibodies reduces apoptosis after irradiation. The hypothesis that an interaction between CD95 and CD95-L is involved in radiation-induced apoptosis is supported by the finding that only peripheral blood lymphocytes that are stimulated with interleukin 2 for 5 days and thus are rendered sensitive to CD95-L show pronounced levels of apoptosis in response to low radiation doses, whereas resting peripheral blood lymphocytes show very low levels of apoptosis after treatment with CD95-L and radiation. The finding that the caspase FLICE is degraded proteolytically after irradiation and the observation of a radiation-induced CD95 receptor capping confirm the functional activation of the CD95 receptor.