OBJECTIVE: The aim of this study is to characterize antigenic sites on HIV-1 gp120 which may be important for the development of active and passive immunization strategies against HIV-1 infection. DESIGN: Two HIV-1-seropositive individuals were selected from the Amsterdam cohort and Epstein-Barr virus (EBV)-transformed B cells were generated from their peripheral blood mononuclear cells, which produce HIV-1-specific human monoclonal antibodies (HuMAb). METHODS: HuMAb were generated and selected based on their reactivities with native gp120. Reactivity with HIV-1 strains from phylogenetically different subfamilies was determined by immunostaining and virus neutralization assays. Specificity for the CD4-binding site was tested by an inhibition enzyme-linked immunosorbent assay and amino acids (aa) involved in the binding of the HuMAb were identified with a set of gp120 molecules with single aa substitutions. RESULTS: Three HuMAb (GP13, GP44, GP68) were generated, all recognizing a conserved conformation dependent epitope within, or topographically near, the CD4-binding site of gp120. HuMAb GP13 and GP68 neutralized a broad range of HIV-1 strains from phylogenetically different subfamilies, whereas HuMAb GP44 exhibited a more restricted pattern of neutralizing activity. The patterns of gp120 aa involved in their binding were unique for each of these HuMAb. CONCLUSIONS: The pattern of reactivities of these three HIV-1-neutralizing HuMAb developed in these studies is similar to, but distinct from other human and rodent MAb that recognize this antigenic site of HIV-1 gp120.