To asses the requirement of interleukin (IL)-10 for peripheral CD4 T cell tolerance, the IL-10 knockout (KO) was introduced into a T cell receptor-transgenic mouse model (TCR1) specific for SV40 T antigen (Tag). IL-10-deficient TCR1-transgenic mice failed to establish antigen-specific T cell tolerance following sequential injections with Tag peptide. Nevertheless, IL-10 was not required for the establishment of CD4 T cell tolerance in double transgenic RT2/TCR1 mice in which Tag is expressed endogenously under control of the insulin promoter. However, in contrast to stable anergy in wild-type RT2/TCR1 mice, tolerant T cells in RT2/TCR1/Il-10KO mice could be driven into vigorous proliferation by exogenous antigenic stimulation in vivo. The observed reactivation of anergic T cell populations in IL-10-deficient mice was only seen after in vivo but not in vitro peptide priming, reflecting an important regulatory function of IL-10 in the context of the living organism. Taken together, these results demonstrate that IL-10 is required to maintain T cell tolerance following exposure to enhanced antigenic stimuli but is not essential for the induction of self-tolerance.