The HIV pandemic continues to pose a global health burden, despite considerable advances in treatment and prevention. Although a large fraction of people with HIV (PWH) are receiving life-saving antiretroviral therapy (ART), treatment does not eradicate the virus. An important co-morbidity relevant to HIV neurologic disease is substance use disorder (SUD), with its negative impact on health by a multitude of factors including injectable drug use, risky behaviors and numerous medical and psychiatric sequelae. Notably, HIV and SUD show a striking neuroanatomical convergence onto the dopamine neuron-rich structures in the ventral midbrain. This includes the ventral tegmental area (VTA) and substantia nigra (SN) with the latter broadly important for mediating habitual behaviors and salience of cues associated with drug use, as well as withdrawal-related anhedonia and dysphoria. However, very little is known about cellular and molecular alterations in the ventral midbrain of PWH with or without SUD co-morbidity, because the majority of relevant studies have been limited to the quantification of neurotransmitter (e.g., dopamine) in bulk tissue and postmortem histology in the context of advanced uncontrolled disease (AIDS). Here, we profile, for the first time, the ventral midbrain transcriptome from HIV+ donors and controls at single (cell) nucleus resolution, and deliver first insights into the cell type-specific neurogenomic response to chronic infection and SUD. We dissected ventral midbrain for unilateral collection of the SN including pars compacta and pars reticulata from n=90 donors collected by the Manhattan HIV Brain Bank (MHBB) from 90 donors (39F/41M; mean age 54.0+/-11.4 years).