The immunoregulatory cytokine interleukin 10 (IL-10) modulates the function of diverse immune and non-immune cells. Here, we examine the chromatin structural changes associated with IL10 gene transcription by naive and differentiated murine T cells. Naive T cells lack DNase I hypersensitive (HS) sites in the vicinity of the IL10 gene, whereas differentiated T cells display a strong 3' constitutive HS site as well as several inducible sites. The majority of HS sites map to regions that are strongly conserved in sequence between mouse and human genomes. In committed Th1 cells, the mechanism of IL10 gene silencing is associated with the development of repressive histone modifications near the IL10 promoter and also near intronic hypersensitive regions of the IL10 gene. Our results constitute the first report of chromatin structural differences within the IL10 gene in differentiated Th1 and Th2 cells and emphasize the surprising diversity of mechanisms used to regulate cytokine gene expression at the chromatin level.