Human B lymphocytes expressing the CD5 surface antigen (CD5+ B cells) constitute a subset capable of producing polyspecific antibodies recognizing a variety of self antigens. The repertoire of antibodies produced by CD5+ and CD5- B cells is different. However, it is not yet established whether this distribution is reflected in different immunoglobulin variable region gene (IgV) use. Rearrangement of heavy chain IgV (IgVH) genes represents one of the first identifiable stages in the maturation of B cells, and occurs in a developmentally ordered fashion. The repertoire of IgVH gene expression is highly restricted during fetal life but diversifies progressively after birth. A high frequency of VH gene use from the relatively small VHIV gene family has previously been demonstrated in human fetal liver B cells. In the present study, 102 B cell lines established by Epstein-Barr Virus-transformation of separated CD5+ and CD5- cord blood B cells, were examined for the frequency of IgV expression using monoclonal antibodies to cross-reactive idiotypes (CRI). The results demonstrate a relatively high frequency of VHIV gene use (30%) in B cells from cord blood. Furthermore, two mutually exclusive CRI associated with distinct subgroups of the VHIV family are segregated in their association with either subset of B cells. One CRI is exclusively expressed in lines established from CD5+ B cells while the other is associated with lines established from CD5- B cells.