As part of the goal of assembling a mixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1 disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epitope can be accessed. From a phage display library derived from an asymptomatic HIV-1 seropositive donor, a panel of recombinant Fabs against the CD4 binding site (CD4bs) of gp120 was retrieved by affinity selection using recombinant gp120 (strain LAI). Two Fabs corresponding to the dominant clones were used to mask the CD4bs epitope(s) before repeating the selection procedure. Four Fabs were then retrieved that had novel heavy chain sequences. Three recognized a novel epitope distinct from that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region (V2 region) dependence indicated by sensitivity to amino acid changes in the V2 loop and by competition with murine anti-V2 mAbs; 2) CD4bs dependence indicated by sensitivity to amino acid changes usually associated with CD4 binding and by inhibition of Fab binding to gp120 by soluble CD4; this dependence seemed to arise via conformational changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a reversal of the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to glycosylated and deglycosylated gp120 and significant, although much reduced, binding to denatured gp120 in contrast with CD4bs Abs, which do not bind to deglycosylated or denatured gp120. One of the novel Fabs efficiently neutralized the MN and LAI strains of HIV-1. These results indicate the presence of a novel neutralizing conformational epitope on gp120 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gp120. The strategy of masking highly immunogenic epitopes with Abs to rescue a broader range of specific Abs from combinatorial libraries should be widely applicable.