OBJECTIVE/DESIGN: The human immunodeficiency virus (HIV) envelope glycoprotein gp120 contributes to neuronal damage that could lead to human immunodeficiency virus-mediated neurocognitive disorder. However, it is debated whether gp120 could promote direct neurotoxicity to synapses because it may not be present in the central nervous system of people living with human immunodeficiency virus (PLWH) on antiretroviral therapy (ART). This study sought to establish whether gp120 is expressed in the human central nervous system in the pre- and post- ART era. METHODS: We utilized real-time polymerase chain reaction (PCR) to detect the envelope mRNA in postmortem caudate nucleus from pre-ART era. HIV negative samples were used as controls. We then used RNAscope fluorescence multiplex to identify which cells express the envelope mRNA. To determine whether gp120 is present despite ART, we analyzed cerebrospinal fluid (CSF) samples from PLWH on ART using mass spectrometry. RESULTS: Using PCR, we detected the envelope mRNA in the caudate nucleus from PLWH with neurocognitive impairment but not in control samples. With RNAscope, we detected the envelope mRNA in microglia and astrocytes in the frontal cortex and caudate nucleus from HIV-positive tissue. With mass spectrometry, we identified a gp120 peptide in the CSF of PLWH on antiretroviral therapy (n = 20) but not in control subjects (n = 7). CONCLUSION: Our data suggest that despite antiretroviral therapy, gp120 is expressed and likely released by infected cells, suggesting that gp120 could be one of the key factors in the neuropathology observed in people living with human immunodeficiency virus.