Haemophilus influenzae b polysaccharide (Hib PS)-protein conjugate vaccines differ chemically and immunologically. To determine whether anti-Hib PS variable region expression might differ according to vaccine formulation, infants were vaccinated at 2, 4, and 6 mo of age with Hib PS coupled to either meningococcal outer membrane protein complex (Hib PS-OMPC) or tetanus toxoid (Hib PS-T), or Hib PS oligomers coupled to a mutant diphtheria toxin (Oligo-CRM). Two anti-Hib PS idiotypes were measured in sera obtained after the third injection: HibId-1, expressed by anti-Hib PS antibodies having the kappa II-A2 variable region, and HibId-2, a newly defined cross-reactive idiotype associated with a subset of anti-Hib PS antibodies having lambda VII variable regions. HibId-1 was present in 33, 68, and 64% of infants given either Hib PS-OMPC, Oligo-CRM, or Hib PS-T, respectively (P < 0.001). The respective values for HibId-2 were 47, 18, and 10% (P = 0.001). Subjects who were vaccinated with Hib PS-OMPC or Hib PS-T and who produced detectable HibId-1-positive antibody, had significantly higher mean antibody avidity than subjects who did not produce HibId-1 positive antibodies. In contrast, Oligo-CRM evoked high avidity anti-Hib PS antibodies, irrespective of the idiotypic profile. These findings indicate fundamental differences in both variable region content and antibody quality elicited by different Hib PS conjugate vaccines.