A considerable part of the Ab repertoire is given over to polyreactive Abs capable of interacting with multiple antigenic species. Neither the function of these Abs nor the molecular basis for their activity is known. To address the latter problem, we have compared the amino acid sequences of a large panel (n = 70) of polyreactive human monoclonal Fab fragments and conducted a series of engineering experiments on a prototype polyreactive Fab. The Fab fragments were retrieved from combinatorial IgG libraries prepared from the bone marrow of long term asymptomatic HIV-1 seropositive donors. The general features displayed by the panel of IgG polyreactive Abs include 1) skewed VH family usage with a predominance of VH1 and VH4 clones and a paucity of the normally prevalent VH3 family; 2) use of a variety of different VH germ-line genes within the context of the family usage and no restriction in D or JH gene usage; 3) skewed VL gene usage: 75% of Fabs used one of two germ lines; and 4) extensive somatic modification of both heavy and light chains. The importance of the heavy chain, in particular the heavy chain CDR3 (HCDR3), in dictating the polyreactive phenotype was demonstrated for the prototype Fab by chain shuffling and CDR transplantation experiments. in addition, and most strikingly, a constrained peptide based on the HCDR3 sequence was shown to be polyreactive and to inhibit binding of the parent Ab to a panel of Ags. A role for conformational flexibility in polyreactivity was suggested by a marked temperature dependence of Ab recognition of Ag. One Ab was shown to be polyreactive at 37 degrees C, but was apparently monoreactive at 4 degrees C. We hypothesize that Ab polyreactivity is associated with conformationally flexible HCDR3 regions in the context of certain favorable framework configurations.