Analysis of VH gene segments deleted in the process of immunoglobulin heavy chain (IGH) variable region assembly in three series of monoclonal B cell lines has been used to determine the human VH region organization. A deletion map of the relative positions of 21 different VH gene segments has been determined. The characterization of B cell lines from three unrelated adults of two racial groups yielded the same relative VH gene segment order, suggesting that the overall order of VH genes in the normal population is constant. This VH gene segment order was consistent with what we had previously generated from physical mapping techniques. DH segments from the second DH cluster, distinct from the major DH locus 3' of the VH region, were not observed to be used in 32 different rearrangements. Approximately 77% of the VH-(D)JH rearrangements involved VH gene segments within 500 kb of the JH region, indicating that human B cell lines preferentially rearrange JH-proximal VH gene segments. The switch, observed in mice, from the fetal use of JH-proximal VH gene segments to an adult VH use dependent upon VH family size may therefore not occur in humans. This detailed map of the VH gene segments is a necessary prerequisite for understanding VH usage in development and disease.