CD4⁺CD25⁺ suppressor T (T_S) cells play a critical role in the maintenance of peripheral tolerance. We examined here proliferative and functional responses as well as differential gene expression in T_S cells. We found that T_S cells were hyporesponsive to antigenic stimuli in vivo and unable to flux Ca²⁺ upon T cell receptor (TCR) engagement. However, T_S cells were not impaired in their proliferative response to lymphopenia, which was dependent on major histocompatibility complex class II expression. Homeostatic proliferation did not abolish T_S cell anergy; rather, it substantially augmented T_S cell function. DNA array analyses identified genes that may inhibit responsiveness at a number of levels in multiple signaling cascades in T_S cells, as well as several anti-apoptotic genes that may mediate their survival.