The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5' end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3' end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.