BACKGROUND.: An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro. METHODS.: Human peripheral blood mononuclear cells (PBMC), CD4T cells, or CD4CD25T cells were stimulated with irradiated human or wild type (WT) or α1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4CD25Tregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xeno-reacitve Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis. RESULTS.: The responses of human PBMC, CD4T cells, and CD4CD25T cells to pig PBMC were stronger than to human PBMC (P<0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P<0.05). Human CD4CD25Tregs suppressed proliferation of CD4CD25T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4CD25FoxP3 responder T cells proliferated more than their xenoreactive counterparts (P<0.05), although xenoreactive CD4CD25T cells proliferated more than alloreactive cells (P<0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4CD25FoxP3 cells. CONCLUSIONS.: Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4CD25FoxP3 cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response. © 2008 by Lippincott Williams and Wilkins.