Pax5 -deficient progenitor B (pro-B) cells are thought to be severely defective for recombination of all immunoglobulin heavy chain (IgH) V gene segments, but the mechanism by which Pax5 regulates this process has not been defined. To address this issue, we have examined the assembly of the IgH locus in Pax5 -deficient pro-B cells and find, unexpectedly, that 3′ IgH V gene segments, which lie closest to the D-J-Cμ region, recombine efficiently, but progressively more distal V gene segments recombine progressively less efficiently. Histone acetylation and germ-line transcription correlate strongly with an open or an accessible chromatin structure thought to be permissive for V(D)J recombination, and defects in recombination are typically accompanied by deficits in these processes. We were therefore surprised to observe that distal V H gene segments in Pax5−/− pro-B cells exhibit no defect in these measures of accessibility. The finding of transcribed, histone acetylated gene segments that fail to recombine suggests that a Pax5 -dependent regulatory mechanism is required in addition to standard constraints of accessibility to control V H gene recombination.