In previous studies of regulation of mouse mixed lymphocyte reactions (MLR) the authors showed that injection of allogeneic spleen cells into footpads activated MLR amplifier T cells in regional lymph nodes and suppressor T cells in spleens. The present studies were performed to determine if anatomic segregation of regulatory T cells was due to antigen induced migration of amplifiers to lymph nodes, or, instead, to inherent differences in regulatory functions of lymph node seeking (LNS) and spleen seeking (SS) T cells. After lethal irradiation, BALB/c and C57Bl/6 mice were injected i.v. with 5 x 107 BALB/c thymocytes. Four days later spleens and lymph nodes were removed from recipients; cells were treated with mitomycin and added to MLR of BALB/c responder cells and C57Bl/6 stimulator cells. Activated BALB/c T cells from spleens of irradiated C57Bl/6 hosts suppressed MLR responses of normal syngeneic spleen cells by 50 to 90%. Conversely, LNS T cells from the same allogeneic hosts enhanced MLR responses 1.5 to 5 fold. When thymocytes were migrated in syngeneic hosts, responses in MLR containing LNS cells were also greater than when SS cells were added, although the magnitude of these differences was less than that observed after migration in allogenic hosts. These data suggest that amplifier and suppressor T cells for mouse MLR are separable subpopulations, with distinctive patterns of migration, and that the regulatory activities of both subpopulations are increased by alloantigen stimulation.