Autoimmune reactivity is a consequence of infection with human immunodeficiency virus (HIV). We studied serological cross-reactions of purified pooled IgG from sera of HIV-infected individuals by using nested sets of synthetic overlapping peptides duplicating the covalent structures of T-cell receptors (TCRs) and immunoglobulin light chains and report that two processes of autoantibody production occur. (i) IgG autoantibodies to putative regulatory variable domain CDR1 and FR3 epitopes (where CDR is complementarity-determining region and FR is framework region) are present in pooled IgG from HIV-infected individuals at levels 10-fold greater than that in pooled IgG from healthy humans. (ii) Anti-TCR autoimmunization involves antigenic mimicry between a conserved peptide stretch of the major neutralizing V3 loop determinant of HIV-1 gp120 and the conserved FR4 segment of the TCR V beta. Affinity-purified antibodies to the synthetic V3 loop peptide bound to a recombinant single-chain TCR and to a synthetic TCR joining segment peptide containing the FR4 sequence. Conversely, affinity-purified autoantibodies from pooled IgG from HIV-infected individuals to the TCR peptide bound the V3 loop peptide and a single-chain TCR. Inhibition studies indicated that the cross-reactive immunizing antigen was the V3 loop. These results bear upon the impact of HIV infection on immune regulation and on the selection of peptides for vaccine development.