Human regulator T (TR) lymphocytes presensitized to allogeneic non-T cells in unidirectional mixed leucocyte cultures (MLC) were shown to be capable of either amplifying or suppressing the MLC proliferative responses of autologous T cells when tested in limiting dilution co-culture assays. Although the concentration of alloantigen present during presensitization influenced the TR suppressive activity, considerable suppression was observed over a 10-20-fold range of stimulators. Thus, the concentration of stimulators was not an extremely critical factor in the generation of TR suppression. Much more important, however, was the duration of MLC presensitization because both TR amplification and suppression of T cell-mediated responses were critically time-dependent. In addition, the deletion of rapidly proliferating T cells from MLC at times of marked amplifier activity resulted in the impaired development of TR suppressor populations. These data directly demonstrate that distinct subpopulations of alloactivated TR cells can be distinguished by varying the presensitization interval and that maximal TR suppression requires earlier proliferation of alloreactive T cells.