We molecularly characterized human immunodeficiency virus type 1 (HIV-1) present in pure populations of astrocytes, macrophages, and multinucleated giant cells isolated using laser capture microdissection from brain tissue of two patients who died with HIV-associated dementia. The V3 region of the HIV-1 envelope (env) gene was amplified from the pure-cell populations, and multiple clones were sequenced. In both patients, the V3 env sequences were distinct in astrocytes compared with neighboring macrophages or multinucleated giant cells and were characteristic of CCR5-using (R5) HIV-1. These results demonstrate cell-specific compartmentalization of distinct R5-like viral strains in the central nervous system microenvironment.