Regulatory T cells (T Reg) control immune responses to self and nonself Ags. The relationship between Ag-driven IL-10-secreting T Reg (IL-10-T Reg) and naturally occurring CD4+ CD25+ T Reg is as yet unclear. We show that mouse IL-10-T Reg obtained using either in vitro or in vivo regimens of antigenic stimulation did not express the CD4+ CD25+ T Reg-associated transcription factor Foxp3. However, despite the absence of Foxp3 expression, homogeneous populations of IL-10-T Reg inhibited the in vitro proliferation of CD4+ CD25− T cells with a similar efficiency to that of CD4+ CD25+ T Reg. This inhibition of T cell proliferation by IL-10-T Reg was achieved through an IL-10-independent mechanism as seen for CD4+ CD25+ T Reg and was overcome by exogenous IL-2. Both IL-10-T Reg and CD4+ CD25+ T Reg were similar in that they produced little to no IL-2. These data show that Foxp3 expression is not a prerequisite for IL-10-T Reg activity in vitro or in vivo, and suggest that IL-10-T Reg and naturally occurring CD4+ CD25+ T Reg may have distinct origins.