The HIV-1 envelope (Env) is the target for neutralizing antibodies and exists on the surface of virions in open or closed conformations. Difficult-to-neutralize viruses (tier 2) express Env in a closed conformation antigenic for broadly neutralizing antibodies (bnAbs) but not for third variable region (V3) antibodies. Here we show that select V3 macaque antibodies elicited by Env vaccination can neutralize 26% of otherwise tier 2 HIV-1 isolates in standardized virus panels. The V3 antibodies only bound to Env in its open conformation. Thus, Envs on tier 2 viruses sample a state where the V3 loop is not in its closed conformation position. Envelope second variable region length, glycosylation sites and V3 amino acids were signatures of neutralization sensitivity. This study determined that open conformations of Env with V3 exposed are present on a subset of otherwise neutralization-resistant virions, therefore neutralization of tier 2 HIV-1 does not always indicate bnAb induction.