We used a TCR-transgenic mouse to investigate whether Th2-mediated airway inflammation is influenced by Ag-specific CD4+ CD25+ regulatory T cells. CD4+ CD25+ T cells from DO11. 10 mice expressed the transgenic TCR and mediated regulatory activity. Unexpectedly, depletion of CD4+ CD25+ T cells before Th2 differentiation markedly reduced the expression of IL-4, IL-5, and IL-13 mRNA and protein when compared with unfractionated (total) CD4+ Th2 cells. The CD4+ CD25−-derived Th2 cells also expressed decreased levels of IL-10 but were clearly Th2 polarized since they did not produce any IFN-γ. Paradoxically, adoptive transfer of CD4+ CD25−-derived Th2 cells into BALB/c mice induced an elevated airway eosinophilic inflammation in response to OVA inhalation compared with recipients of total CD4+ Th2 cells. The pronounced eosinophilia was associated with reduced levels of IL-10 and increased amounts of eotaxin in the bronchoalveolar lavage fluid. This Th2 phenotype characterized by reduced Th2 cytokine expression appeared to remain stable in vivo, even after repeated exposure of the animals to OVA aerosols. Our results demonstrate that the immunoregulatory properties of CD4+ CD25+ T cells do extend to Th2 responses. Specifically, CD4+ CD25+ T cells play a key role in modulating Th2-mediated pulmonary inflammation by suppressing the development of a Th2 phenotype that is highly effective in vivo at promoting airway eosinophilia. Conceivably, this is partly a consequence of regulatory T cells facilitating the production of IL-10.