Several regulatory regions are important for the expression of genes encoding T helper type 2 (T(H)2) cytokines, including T(H)2-specific DNase I hypersensitivity sites in the T(H)2 cytokine locus control region. Among these sites, Rad50 hypersensitive site 7 (RHS7) shows rapid T(H)2-specific demethylation after antigenic stimulation. To investigate the function of RHS7 in T(H)2 cell differentiation, we have generated RHS7-deficient mice. CD4(+) T cells and mast cells showed a notable reduction in T(H)2 cytokine expression in vitro and T(H)2 responses in vivo were considerably impaired in RHS7-deficient mice. Deletion of RHS7 did not affect the expression of a linked Rad50 gene, but it did reduce long-range intrachromosomal interactions between the locus control region and promoters of the T(H)2 cytokine genes. Our findings show that RHS7 is essential for the proper regulation of T(H)2 cytokine gene expression.