The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Eμ) only modestly reduce D H→JH rearrangements when assessed in peripheral B cells. However, deletion of Eμ severely impairs recombination of V H gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Eμ deletions on primary DH→JH rearrangement: 1) Accessibility at the D HJH cluster is controlled by a redundant cis-element in the absence of Eμ. One candidate for this element lies 5′ to D Q52 (PUQ52) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, DH→J H recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PDQ52 and Eμ in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PDQS2 promoter is dispensable for germline transcription and recombination of the DHJH cluster. In contrast, we demonstrate that Eμ directly regulates accessibility of the DHJH region. These findings reveal a significant role for Eμ in the control mechanisms that activate IgH gene assembly and suggest that impaired VH→D HJH rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in DH→JH recombination.