Reactive astrocytosis is a well-documented feature of HIV encephalitis (HIVE), but it is unclear whether restricted infection of astrocytes contributes to this phenomenon. In addition, the part played by reactive and/or infected astrocytes in AIDS-related dementia is not fully understood. In this study of patients at different stages of the human immunodeficiency virus (HIV) infection, who had been treated at most with one antiretroviral drug, reactive astrocytes were identified by immunopositivity for glial fibrillary acidic protein (GFAP) and infected astrocytes by positivity for HIV Nef protein. Results were compared for drug-using AIDS patients with (n=9) and without (n=7) HIVE, for presymptomatic HIV-positive drug users (n=12) and for control HIV-negative subjects (n=20), including a group who used drugs (n=10). GFAP-reactive astrocytes in both grey and white matter were significantly more numerous in HIVE subjects than in each of the other groups but did not correlate with viral load. Nef-positive astrocytes were confined to HIVE cases and to white matter, but were numerous in only one subject who was treatment-naive. Nef-positive microglia were identified in all HIVE cases and in occasional AIDS and presymptomatic subjects who did not have HIVE. The results suggest that astrocytes may form an additional viral reservoir in late HIV infection and may contribute to HIVE. However, the number of GFAP-positive astrocytes was neither increased in pre-AIDS nor in drug abuse, in contrast with microglia which we have shown previously to be up-regulated in both states.