Neutralizing antibodies specific for the third variable (V3) domain of gp120, the HIV-1 surface envelope protein, appear early in infection. However, they are usually highly specific for the priming isolate. To identify potential mimotopes of the V3 domain, we have screened a hexapeptide phage library with a human neutralizing mAb, mAb 268, specific for the V3 loop of the viral MN isolate. We have identified two groups of sequences. Within the first group, sequence 268-1 reproduces the linear epitope identified using a conventional epitope mapping approach. The sequence 268-1, H L G P G R, corresponds to amino acids 315-320, localized in the highly conserved tip of the V3 loop. A second group of sequences was identified, including sequence 268-2, K A I H R I. Partial homology with a more variable region of the V3 loop can be found. Using synthetic peptides, we demonstrated that peptides, 268-1 and 268-2, both interact with the same binding site as the V3 region on the 268 mAb. Moreover, both peptides can inhibit the interaction of the 268 mAb with the original immunogen, gp120MN. Peptide 268-1 can compete with peptide 268-2, albeit poorly, for binding of the 268 mAb. When injected into rabbits, KLH conjugated peptide 268-2 elicited antibodies that interact specifically with the initial immunogen gp120MN. These data suggest that peptide 268-2 is both an antigenic and immunogenic mimic of the natural antigen, gp120MN.