Re-exposure of day 14 mixed leukocyte culture (MLC) cells to the original stimulating alloantigens (secondary response) has previously been shown to result in significant proliferation and in rapid reappearance of high levels of cytolytic T-lymphocyte (CTL) activity within the next 4 days. Moreover, evidence has been presented that CTL precursor cells in day 14 MLC populations, while they derived from cells were large at peak of the primary response (day 4) were themselves small lymphocytes which developed into large CTL after restimulation. In this study, inhibition of DNA synthesis by cytosine arabinoside (ARA-C) was used to investigate whether CTL formation could be dissociated from proliferation during the secondary response. It was found that within the first 24 h after restimulation (a) CTL activity increased 6-to-20-fold, (b) 60-70% of the small T lymphocytes became medium- to large-sized cells, and (c) both events were independent of DNA synthesis. By using two successive cell separations by velocity sedimentation at unit gravity, before and after stimulation of day 14 MLC cells for 24 h in the presence or absence of ARA-C, direct evidence was obtained that small CTL precursor cells developed into large CTL, irrespective of DNA synthesis. The presence of ARA-C for periods longer than 24 h inhibited any further increase in CTL activity, in contrast to a parallel increase in lytic activity and cell number from day 1 to day 4 in control restimulated cultures. Taken together with the finding that 90% of the medium- and large-sized lymphoid cells in control restimulated cultures underwent DNA synthesis within 24 h, these results thus suggest that during a secondary MLC response there is initially a differentiation step leading to the formation of CTL which, although it can be clearly dissociated from DNA synthesis, is under normal conditions followed by proliferation of these effector cells.