HIV infection induces antibodies that mediate neutralization of cell-free virus and may protect against viral infection. Neutralizing human mAb that bind to the third hypervariable domain (V3) of gp120 have been generated from PBL of HIV-seropositive subjects. Twelve such mAb recognize 9 different epitopes spanning an 11 amino acid stretch at the tip of the V3MN loop. The epitopes of an additional two mAb have not been precisely determined but occur within a 15-mer peptide around the tip of the V3 loop. Eleven of 13 mAb are reactive with at least one other V3 peptide besides MN, indicating that cross-reactivity is a common phenomenon amongst anti-V3 antibodies. All the mAb achieved 50% neutralization of HIVMN at antibody concentrations of 12 to 4700 ng/ml. Two mAb, which recognized epitopes at the top of the V3 loop, GPGR and GRAF, neutralized strains as divergent as MN and IIIB. The affinities of all mAb tested were shown to be substantially higher for the rgp120 than for a 23-mer peptide from the V3 loop of the homologous strain. A significant inverse correlation was demonstrated between affinities and the 50% neutralizing doses for HIVMN.