This study determined the distribution of in situ polymerase chain reaction (PCR)-amplified HIV-1 nucleic acids in the central nervous system (CNS). Amplified viral DNA was detected in each of the seven HIV-1-positive cases and in none of the seven negative controls. HIV-1 DNA was rarely detected with standard in situ hybridization, consistent with low levels of proviral DNA. In patients with minimal clinical and pathological CNS involvement, only rare HIV-1 DNA-positive perivascular microglial cells were noted. In patients with dementia, many infected neurons and astrocytes as well as microglial cells were detected. Severe disease was also characterized by the detection of tumor necrosis factor-alpha (TNF-alpha) mRNA and viral RNA by reverse transcription (RT) in situ PCR. These results suggest that HIV-1 commonly exists in the CNS in the asymptomatic patient and that progression is marked by a dramatic increase of the number of cells with HIV-1 DNA, including neurons and astrocytes, and a concomitant upregulation of both viral and TNF-alpha transcription.