A procedure has been previously developed to allow kinetic analysis of target cell lysis in individual conjugates formed between murine alloimmune peritoneal exudate lymphocytes (PEL) and relevant tumor cells in suspension. Under the conditions used in the present work, 60% of the conjugates consisted of one lymphocyte bound to one target cell, whereas the remaining were multicellular, i.e., contained one lymphocyte bound to either two (24%), three (12%), or four (4%) target cells. Both ultrastructural studies and micromanipulation experiments with individual multicellular conjugates indicated differences in the degree of target cell binding to the same PEL. In more than 80% of the multicellular conjugates, lysis of all conjugated target cells occurred within a 3-hr incubation period at 37°C. Kinetic analysis of target cell lysis, however, showed that the time required for target cell death was quite variable, both within the same multicellular conjugate and between different conjugates, ranging from a few minutes to 3 hr after conjugation. By comparing the elapsed time necessary for lysis of 50% of the target cells in conjugates containing one lymphocyte bound to three target cells, it could be estimated that, on the average, lysis of the first, second, and third target cells occurred after 30, 60, and 90 min, respectively. The time required for lethal hit of each conjugated target cell was measured by adding EDTA to the incubation medium of individual multicellular conjugates at various time intervals. Evidence could be obtained that a CTL induced irreversible lesions in only one of the conjugated target cells at a time, i.e., lethal hits were given sequentially rather than simultaneously.