Allergy is associated with pathological Th2 responses to otherwise harmless environmental Ags. In contrast, nonallergic individuals mount nonpathological immune responses to allergens, partly attributed to regulatory T cell (Treg) activity. Although thymus-derived natural Tregs have been shown to maintain tolerance to self-Ags and prevent autoimmunity, the generation of Tregs specific to non-self-Ags is less well understood. We investigated the potential for induction of Tregs from PBMCs of ryegrass pollen-allergic or healthy subjects by stimulation in vitro with ryegrass pollen extract in the absence of additional exogenous stimuli. We found that two subsets of proliferating CD4(+) T cells were induced, one expressing intermediate levels of Foxp3 (and IFN-gamma, IL-4, IL-17, or IL-2) and the other expressing high levels of Foxp3 (and no effector cytokines). After enrichment based on CD39 expression, the Foxp3(hi) subset suppressed CD4(+) T cell proliferation and IFN-gamma production. The Foxp3(hi) Treg originated from both conversion of dividing non-Tregs (CD4(+)CD25(-)CD127(hi)) and expansion of natural Tregs (CD4(+)CD25(+)CD127(lo)). Stable functional Tregs expressing high levels of Foxp3 were induced simultaneously with effector T cells by allergen stimulation. Induction of Foxp3(hi) Tregs was reduced in allergic subjects. These results indicate that the cogeneration of Foxp3(hi) Tregs in response to allergen may be a mechanism for controlling allergic reactions in healthy individuals, which is impaired in those with allergies.