We studied the effects of alemtuzumab on T-regulatory cells (Tregs) during alloactivation, first by differences in depletion of various naive versus alloactivated cell subsets in peripheral blood of healthy volunteers, then by adding serial concentrations to human leukocyte antigen (HLA)-DR-matched and -mismatched responding and stimulating cells in mixed lymphocyte reaction (MLR). Lymphoproliferation inhibition and the development of proliferating carboxyfluorescein succinimidyl ester (CFSE)-diluted CD4(+)CD25(high)CD127(-)FOXP3(+) (phenotypic) Tregs by flow cytometry were measured. Also, the ability of alemtuzumab-treated versus nontreated MLR generated CD4(+)CD127(-) cells to allospecifically inhibit MLRs and recruit additional responding Tregs was tested. We found a more pronounced refractoriness of alloactivated versus naive CD4(+)CD25(high) cells to alemtuzumab induced lymphodepletion. Alemtuzumab dose dependently inhibited lymphoproliferation while amplifying percentages of MLR-generated Tregs. This was somewhat augmented by human complement addition. CD127(-)CD4(+) cells immunoselected after 7 days from alemtuzumab-treated MLRs allospecifically inhibited lymphoproliferation and recruited additional Tregs in fresh MLR-responding cells, similar to modulators derived from MLRs without drug addition (media). Addition of tacrolimus and sirolimus to alemtuzumab further inhibited MLR proliferation. However, Treg percentages were markedly higher with sirolimus. These results support the notion that alemtuzumab induces immunoregulation in naïve T cells undergoing alloactivation absent presensitization, especially used in conjunction with maintenance SRL.