The CD4+CD25+ regulatory T cells (Tregs) are efficient regulators of autoimmunity, but the mechanism remains elusive. We summarize recent data for the conclusion that disease-specific Tregs respond to tissue antigens to maintain physiological tolerance and prevent autoimmunity. First, polyclonal Tregs from antigen-positive donors suppress autoimmune ovarian disease (AOD) or experimental autoimmune prostatitis in day 3 thymectomized (d3tx) mice more efficiently than Tregs from antigen-negative donors. Second, Tregs of antigen-negative adult mice respond to cognate antigen in vivo and rapidly gain disease-specific Treg function. Third, in d3tx female recipients devoid of neonatal ovarian antigens, only female Tregs suppressed AOD; the male Tregs gain AOD-suppressing function by responding to the ovarian antigen in the recipients and mask the supremacy of female Tregs in AOD suppression. Fourth, when Tregs completely suppress AOD, the ovary-draining lymph node is the only location with evidence of profound and persistent (but reversible) host T-cell suppression. Fifth, from these nodes, highly potent AOD-suppressing Tregs are retrievable. We conclude that self-tolerance involves the continuous priming of Tregs by autoantigens, and in autoimmune disease suppression, the effector T-cell response is continuously negated by potent disease-specific Tregs that accumulate at the site of autoantigen presentation.