Human immunodeficiency virus (HIV) and methamphetamine (METH) use disorders are associated with cerebral dysfunction. To determine whether these effects were evident on in vivo neuroimaging, quantitative, single voxel magnetic resonance (MR) spectroscopy was used to assess frontal white matter, frontal gray matter, and basal ganglia in 40 HIV+/METH+, 66 HIV+/METH-, 48 HIV-/METH+, and 51 HIV-/METH- participants. HIV was associated with lower N-acetylaspartate (NAA) in frontal white and frontal gray matter but METH was not associated with cerebral metabolite differences in any region. Among HIV+ individuals, lower CD4 counts and higher plasma HIV viral loads were associated with lower NAA in frontal gray matter and basal ganglia. The relationship between detectable plasma HIV viral load and NAA in frontal white matter was significantly stronger in the HIV+/METH+ group, compared to HIV+/METH-. Higher detectable plasma HIV viral load was significantly associated with higher myo-inositol (MI) in frontal white and gray matter for HIV+/METH+, but not HIV+/METH-. For the HIV-/METH+ group, lifetime duration of METH use was associated with higher choline levels in frontal gray matter and higher MI levels in basal ganglia. Our findings are consistent with significant disruption of neuronal integrity in the frontal lobes of HIV-infected individuals. Although METH was not associated with cerebral metabolite levels, other findings suggested that METH use did affect the brain. For example, the relationship between detectable plasma HIV viral load and NAA levels was limited to HIV+/METH+ individuals. This evidence indicates when HIV is poorly suppressed, METH may modify the effects of the virus on neuronal integrity.