HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates. •In lab-adapted tier-1 Env isolates, the Env CoRbs is directly accessible to CD4-induced (CD4i) antibodies.•Inaccessibility of the Env CoRbs in tier-2 & tier-3 isolates is associated with lack of neutralization by CD4i antibodies.•Lower molecular mass CD4i antibody domains such as m36.4 can access the CoRbs.•M36.4 neutralizes some tier-2 and tier-3 Env isolates even prior to engagement to the primary receptor, CD4.•Neutralization of cell-free viruses by m36.4 is Env-specific and independent of viral subtype or tier categorization.