Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.