Sera from individuals infected by HIV-1 usually neutralize multiple viral isolates. To determine the extent to which these neutralizing antibodies recognize a principal neutralizing determinant in the V3 region of the envelope protein gp120 (amino acids 308-332), one broadly neutralizing serum was fractionated by affinity chromatography on immobilized peptide columns. Antibodies that neutralize one isolate (HTLV-IIIMN) were substantially but not completely absorbed by the peptide corresponding to a portion of its V3 determinant, whereas the antibodies that neutralize two other isolates (HTLV-IIIB and HTLV-IIIRF) were not absorbed by homologous peptides corresponding to their neutralizing determinants. Neutralizing antibodies also failed to be absorbed by full length envelope protein gp160 and by two other envelope peptides previously reported to be broadly neutralizing epitopes (amino acids 254-274 and 735-752). We conclude that the infected individual had raised a type-restricted neutralizing response targeted at a linear epitope in the V3 region, and that broad neutralization resulted from recognition of epitopes not yet identified.