To better understand the limits of antigenic reactivity and epitope accessibility of the V3 domain of primary HIV-1 isolates, we evaluated three human anti-V3 monoclonal antibodies (mAbs) and selected guinea pig vaccine sera for neutralization against reference panels of subtype B and C pseudoviruses derived from early stage infections. The mAbs and vaccine sera potently neutralized several prototype viruses, but displayed substantially less neutralization of most reference strains. In the presence of soluble CD4 (sCD4), the breadth of V3-mediated neutralization was increased; up to 80% and 77% of the subtype B and C viruses respectively were sensitive to V3-mediated neutralization. Unlike sCD4, the reaction of CD4-binding site mAbs b12 and F105 with native virus did not lead to full exposure of the V3 domain. These findings confirm that V3 antibodies recognize most primary viral strains, but that the epitope often has limited accessibility in the context of native envelope spike.