A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4(+)CD25(+)FOXP3(+) natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS(-/-) mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS(-/-) nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS(-/-) nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.