The transcriptional coactivator CIITA regulates MHC class II genes. In the mouse, CIITA is expressed from three distinct promoters (pI, pIII, and pIV) in a developmental and cell type-specific manner with pIII being responsible for B lymphocyte-specific expression. Although the promoter proximal sequences that regulate CIITA in B cells have been described, nothing is known about additional distal elements that may regulate its expression in B cells. Sequence homology comparisons, DNase I hypersensitivity assays, and histone modification analysis revealed a potential regulatory element located 11 kb upstream of pIII. Deletion of this element, termed hypersensitive site 1 (HSS1), in a bacterial artificial chromosome encoding the entire CIITA locus and surrounding genes, resulted in a complete loss of CIITA expression from the bacterial artificial chromosome following transfection into B cells. HSS1 and pIII displayed open chromatin architecture features in B cell but not in plasma cell lines, which are silenced for CIITA expression. PU. 1 was found to bind HSS1 and pIII in B cells but not in plasma cells. Depletion of PU. 1 by short hairpin RNA reduced CIITA expression. Chromatin conformation capture assays showed that HSS1 interacted directly with pIII in B cells and that PU. 1 was important for this interaction. These results provide evidence that HSS1 is required for B cell-specific expression of CIITA and that HSS1 functions by interacting with pIII, forming a long-distance chromatin loop that is partly mediated through PU. 1.