The 3' regulatory region (3' RR) of the murine immunoglobulin heavy chain (IgH) locus contains multiple DNase I-hypersensitive (hs) sites. Proximal sites hs3A, hs1.2, and hs3B are located in an extensive palindromic region and together with hs4 are associated with enhancers involved in the expression and class switch recombination of IgH genes. Distal hs5, -6, and -7 sites located downstream of hs4 comprise a potential insulator for the IgH locus. In pro-B cells, hs4 to -7 are associated with marks of active chromatin, while hs3A, hs1.2, and hs3B are not. Our analysis of DNA methylation-sensitive restriction sites of the 3' RR has revealed a similar modular pattern in pro-B cells; hs4 to -7 sites are unmethylated, while the palindromic region is methylated. This modular pattern of DNA methylation and histone modifications appears to be determined by at least two factors: the B-cell-specific transcription factor Pax5 and linker histone H1. In pre-B cells, a region beginning downstream of hs4 and extending into hs5 showed evidence of allele-specific demethylation associated with the expressed heavy chain allele. Palindromic enhancers become demethylated later in B-cell differentiation, in B and plasma cells.