The present study demonstrates that CD4⁺CD25⁺ T cells, expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART), exhibit phenotypic, molecular, and functional characteristics of regulatory T cells. The majority of peripheral CD4⁺CD25⁺ T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4⁺CD25⁺ T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4⁺CD25^– T cells, CD4⁺CD25⁺ T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin, cytomegalovirus (CMV), and p24 significantly increased following depletion of CD4⁺CD25⁺ T cells. Furthermore, addition of increasing numbers of CD4⁺CD25⁺ T cells resulted in a dose-dependent inhibition of CD4⁺CD25^– T-cell proliferation to tuberculin and p24. CD4⁺CD25⁺ T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor β (TGF-β) and interleukin 10 (IL-10), thus indicating the presence of p24-specific CD4⁺ T cells among the CD4⁺CD25⁺ T-cell subset. Suppressive activity was not dependent on the secretion of TGF-β or IL-10. Taken together, our results suggest that persistence of HIV antigens might trigger the expansion of CD4⁺CD25⁺ regulatory T cells, which might induce a tolerance to HIV in vivo.