Astrocyte activation has been postulated to be a major contributor to functional changes in the brain of AIDS patients. We assessed astrocyte activation in the simian immunodeficiency virus (SIV) model. Four groups of macaque brains were examined: uninoculated controls, animals inoculated with virus that did not cause disease, animals inoculated with virus that caused AIDS but did not cause encephalitis, and animals with SIV encephalitis. We examined expression of calbindin-D-28K, a calcium binding protein that is upregulated in astrocytes during excitotoxic events, as well as glial fibrillary acidic protein (GFAP). The presence of calbindin in astrocytes was confirmed by double-labeling using confocal microscopy. Increases in calbindin staining were most apparent in the white matter, but increases in GFAP staining were most apparent in middle layers of the cerebral cortex. Six of the seven animals with SIV encephalitis had calbindin immunoreactive astrocytes in the subcortical white matter, corpus callosum, internal capsule, cerebral peduncle, pontine white matter, and cerebellar white matter. Very rarely, a few, very lightly calbindin-immunoreactive astrocytes were present in the uninoculated control brains. The increase in calbindin expression by astrocytes in SIV encephalitis suggests that these cells are subject to calcium toxicity. In uninoculated control macaques, and in macaques inoculated with virus that did not cause disease, GFAP-immunoreactive astrocytes were present throughout the subcortical white matter and in layer I, but very few were found in layers III-V of the cerebral cortex. Two animals that died of AIDS without encephalitis had somewhat higher numbers of GFAP immunoreactive astrocytes in middle cortical layers. In seven animals that received passaged neurovirulent virus and developed both AIDS and encephalitis, the number of GFAP-immunoreactive astrocytes in middle cortical layers was high, indicating widespread astrocyte activation.