CD3- and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens. To investigate whether this impaired alloreactivity is a result of preferential expansion of regulatory T (Treg) cells, we compared peripheral blood mononuclear cells (PBMC) activated with CD3 and CD28 antibodies co-immobilized on beads and expanded for 12 days with interleukin (IL)-2 (Co(CD3/CD28) cells) to the respective unactivated PBMC in terms of proliferation, cytokine production, and expression of Treg markers [cytotoxic T-lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor (GITR) and forkhead box P3 (FoxP3)] after allostimulation. Alloreactive cells were identified by carboxyfluoresceine succinimidyl ester staining dilution. Alloreactive cells in Co(CD3/CD28) cells had a lower proliferative response and a lower potential for IL-2 and interferon-gamma secretion than did those in PBMC, demonstrating a functional impairment of alloreactive cells during ex vivo expansion. Expression of Treg markers transiently increased during ex vivo expansion and was unaffected by depletion of CD25(+) cells (containing Treg cells) before ex vivo PBMC expansion. Such prior CD25(+) depletion did not restore the alloreactivity of Co(CD3/CD28) cells. After allostimulation, expression of Treg markers was restricted to proliferative (alloreactive) cells among PBMC or Co(CD3/CD28) cells. Lastly, CD4(+) CD25(+) cells purified from Co(CD3/CD28) cells lacked suppressive activity when used as a third party, in contrast to CD4(+) CD25(+) cells purified from PBMC. In conclusion, the impaired alloreactivity of T cells expanded ex vivo is not a result of preferential Treg cell expansion and/or enhanced suppressive Treg activity.