Total anti-gpl20 antibodies (total anti-gpl20 Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120sF2-Sepharose column and exhibited both type-and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutrazing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gpl20 Abs by using a CD4-blocked gpl2OsF2-Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting typeand group-specific neutralizing activities of total anti-gpl20 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-speciflc antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gpl20 antibodies are specific for the CD4 attachment site.Although the precise role of the third variable region (V3) of gpl20 in virus infection is still in question, antibodies to this region elicited by vaccination or infection neutralize virus infectivity by interfering with a postbinding event (1-4). Furthermore, antibodies to this region exert their primary neutralizing effects on the virus strain used in immunization (5-8) and have been called "type-specific neutralizing antibodies." Under some circumstances, cross-neutralizing antibodies to the V3 domain have been observed (9). The hypervariability of the envelope region outside as well as within the V3 domain suggests that HIV mutants emerge over time that escape V3 type-specific neutralizing antibodies (10, 11). Primary, secondary, and tertiary structure of the V3-region molecule are important in antibody binding and neutralization (12, 13). Since all known HIV isolates retain the ability to infect CD4+ cells despite considerable sequence variation within gpl20, it has been suggested that the CD4 attachment site on gp120 consists of conserved regions (14,15). Antibodies binding to this CD4 attachment site are expected to block infection of a majority (if not all) of HIV isolates by interfering with the interaction with CD4+ cells. Antibodies specific against this region have been detected in the sera ofHIV-infected humans (16)(17)(18). A recent report (19) described a human monoclonal antibody derived from a seropositive patient that is specific for the CD4 attachment site and neutralizes multiple HIV isolates. To date no attempts have been made to our knowledge to purify these antibodies and directly correlate their activity and concentrations associated with neutralization of multiple strains of HIV-1.This study was undertaken to gain a better understand...