Interleukin-10 (IL-10), an immunoregulatory cytokine, modulates the function of various immune and nonimmune cells, yet little information is available on the molecular mechanism of transcriptional regulation at the chromatin level. During T cell differentiation from naive T cells into Th1 and Th2 cells, the expression of IL-10 in Th1 cells slowly disappears, whereas Th2 cells produce more IL-10. We examined the chromatin structural changes associated with IL-10 gene transcription by naive and differentiated murine Th1 and Th2 cells. Naive T cells lack DNase I hypersensitivity (HS) sites in the vicinity of the IL-10 gene, whereas differentiated T cells display a strong 3' constitutive HS site as well as several inducible sites. In committed Th1 cells, the mechanism of IL-10 gene silencing is associated with a closed chromatin structure, the lack of an HS site at the promoter region, and the development of repressive histone modification near the IL-10 promoter and introns 3 and 4. We confirm that the majority of HS sites coincide with conserved noncoding sequences (CNSs) identified by comparative genomic sequence alignment between human and mouse genomes. Potential transcription factor binding sites were located by comparing CNSs with the TRANSFAC database. Predicted in vivo binding of specific factors on the CNS locus were confirmed by chromatin immunoprecipitation assays. Our results suggest that the combination of HS site and comparative genomic approaches allows identification of regulatory elements involved in differential IL-10 gene expression between Th1 and Th2 cells during T cell differentiation.