Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.