The antibody response to Haemophilus influenzae type b polysaccharide (Hib PS) is known to be encoded by a few V-region genes. We have obtained four human monoclonal Hib PS antibodies from four healthy adult subjects immunized with diphtheria toxin-conjugated Hib PS vaccine. The VH gene segments that encode for these antibodies belong to the VH3 gene family, of which two are related to the V3-23 gene and two to the VH3b subfamily. Both hybridomas that express a V3-23-related gene use short D-segments (3 bp), the JH6 gene segment and a V kappa gene derived from the A2 germline gene. The two hybridomas that express VH3b genes use D-segments of conventional length (24-33 bp), the JH4 gene segment and a non-A2 V kappa gene. Comparison of our sequences with those reported by others suggests that the above patterns of V-region gene segment association exemplify two V-region gene configurations that are predominant in the Hib PS antibody response. The first configuration is reminiscent of antibodies produced by B-1 B cells while the second is more characteristic of antibodies produced by conventional B cells. The possibility that these two configurations, in fact, represent the products of two different B cell lineages remains to be elucidated.