To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the V(H)III family. In contrast, the L chains of these antibodies were clearly from at least four different V(L) families; V(K)I, V(K)II, V(K)III, and V(λ). Interestingly, V(L) family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. V(K)II antibodies did not cross-react, whereas antibodies expressing V(λ), V(K)I, or V(K)III generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.