We have devised a simple assay that provides an instantaneous representation of VH family usage in primary and peripheral lymphoid tissues. This assay lacks complex manipulations out of the animal and thus minimizes the risk of in vitro artifacts. We have used this assay to demonstrate a dramatic preference for utilization of the most JH-proximal VH segments in the newborn liver of BALB/c and C57BL/6 mice. Furthermore, we find that VH segments from across the entire VH locus are utilized early in development, but at frequencies directly related to their JH proximity. A major shift away from the position-dependent VH repertoire of the neonate is seen in unprimed or polyclonally-activated adult spleen cells, in which relative utilization of the various VH families is related to family size. We also report consistent strain-specific differences in the expression of certain VH families. Our data indicate that a position-dependent VH repertoire is generated in differentiating pre-B lymphocytes (probably reflecting constraints imposed by the immunoglobulin gene assembly process), and that mechanisms that operate subsequent to rearrangement then randomize this position-dependent repertoire in a strain-specific manner.