Previous studies have demonstrated that murine CD4 Th1 cells lack perforin and use a pathway distinctive from CD8 CTL to express cytotoxicity. Whether the cytotoxic process of Th1 cells can be separated into identifiable stages and how these differences affect this process were determined in this study. We have resolved the cytotoxic process of Th1 clones into three stages identical with those of CD8 CTL, namely, conjugate formation/activation, lethal hit. and effector-independent programming for target DNA fragmentation. By comparing the cytotoxic processes between Th1 clones on Ag-pulsed targets and (PMA+A23187)-activated Th1 clones on unpulsed targets, we have also demonstrated that 1) the requirement of CD4 Th1 cells for de novo synthesis of cytotoxic machinery was partly responsible for the lag time in the induction of target DNA fragmentation by Th1 clones; 2) lethal hit was delivered rapidly; 3) lethal hit under forced contact by centrifugation did not need extracellular Ca2+ and Mg2+; 4) without centrifugation, lethal hit required extracellular Mg2+, but not Ca2+; 5) the average functional half life of the cytotoxic machinery was 54 +/- 24 (n = 4) min. The data demonstrate that the cytotoxic process of Th1 clones uses an activation-dependent cytotoxic machinery to deliver a short-lived, short-ranged, and quick-acting lethal hit to target, which induces a program in target for DNA fragmentation.