In HIV‐1 encephalitis, HIV‐1 replicates predominantly in macrophages and microglia. Astrocytes also carry HIV‐1, but the infection of oligodendrocytes and neurons is debated. In this study we examined the presence of HIV‐1 DNA in different brain cell types in 6 paraffin embedded, archival post‐mortem pediatric and adult brain tissues with HIV‐1 encephalitis by Laser Capture Microdissection (LCM). Sections from frontal cortex and basal ganglia were stained by immunohistochemistry for CD68 (microglia), GFAP (astrocytes), MAP2 (neurons), and p24 (HIV‐1 positive cells) and different cell types were microdissected by LCM. Individual cells or pools of same type of cells were lysed, the cell lysates were subjected to PCR using HIV‐1 gag SK38/SK39 primers, and presence of HIV‐1 DNAwas confirmed by Southern blotting. HIV‐1 gag DNAwas consistently detected by this procedure in the frontal cortex and basal ganglia in 1 to 20 p24 HIV‐1 capsid positive cells, and in pools of 50 to 100 microglia/macrophage cells, 100 to 200 astrocytes, and 100 to 200 neurons in HIV‐1 positive cases but not in HIV‐1 negative controls. These findings suggest that in addition to microglia, the infection of astro‐cytes and neurons by HIV‐1 may contribute to the development of HIV‐1 disease in the brain.