The T helper type 2 (Th2) cytokine genes Il4, Il5, and Il13 are contained within a 140-kb region of mouse chromosome 11 and their expression is controlled by a locus control region (LCR) embedded within this locus. The LCR is composed of a number of DNase I-hypersensitive sites (HSs), which are believed to encompass the regulatory core of the LCR. To determine the function of these sites, mutant mice were generated in which combinations of these HSs had been deleted from the endogenous LCR, and the effect on Th2 cytokine expression was assessed through the use of in vivo and in vitro models. These experiments revealed that, although all of the hypersensitive sites analyzed are important for appropriate LCR function, some sites are more important than others in regulating cytokine expression. Interestingly, each LCR mutation showed contrasting effects on cytokine expression, in some cases with mutants displaying opposing phenotypes between in vitro cultures and in vivo immunizations. These studies indicated that Rad50 hypersensitive site 6 was the singularly most important HS for Th2 cytokine expression, displaying consistent reductions in cytokine levels in all models tested. Furthermore analysis of chromatin modifications revealed that deletion of Rad50 hypersensitive site 6 impacted epigenetic modifications at the promoters of the Il4, Il5, and Il13 genes as well as other regulatory sites within the Th2 locus.