To investigate the independent and interactive effects of opiate addiction and HIV on neuroinflammation, we measured microglial/macrophage activation and astrogliosis in multiple regions of human brain. Samples of thalamus, frontal gray matter, and frontal white matter were obtained from 46 individuals categorized as: HIV negatives, HIV-negative opiate addicts, HIV positives, HIV-positive opiate addicts, HIV encephalitis (HIVE), and HIVE opiate addicts. Activated brain microglia/macrophages and astrocytosis were quantified by morphometric analysis of immunohistochemical stains for CD68, HLA-D, CD163, and GFAP. The effects of HIV grouping, opiate addiction, and their interaction on expression of the markers were examined in a series of two-way ANOVAs. In opiate addicts, there was generally higher baseline expression of CD68 and HLA-D in HIV negatives, and lower expression in HIV and HIVE, compared to individuals without opiate abuse. Thus, for these markers, and for GFAP in frontal gray, opiates were associated with attenuated HIV effect. In contrast, for CD163, opiates did not significantly alter responses to HIV, and HIV effects were variably absent in individuals without opiate abuse. The divergent impact that opiate addiction displays on these markers may suggest a generally immunosuppressive role in the CNS, with decreased HIV-associated activation of markers CD68 and HLA-D that potentially reflect neurotoxic pathways, and preservation of CD163, thought to be an indicator of neuroprotective scavenger systems. These results suggest a complex impact of opiates on neuroinflammation in baseline and virally stimulated states.