The failure of CD25⁺ regulatory T cells (T_regs) to proliferate after T cell receptor (TCR) stimulation in vitro has lead to their classification as naturally anergic. Here we use T_regs expressing a transgenic TCR to show that despite anergy in vitro, T_regs proliferate in response to immunization in vivo. T_regs also proliferate and accumulate locally in response to transgenically expressed tissue antigen whereas their CD25⁻ counterparts are depleted at such sites. Collectively, these data suggest that the anergic state that characterizes CD25⁺ T_regs in vitro may not accurately reflect their responsiveness in vivo. These observations support a model in which T_reg population dynamics are shaped by the local antigenic environment.