Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.