Objective: The presence of HIV-1 in postmortem brain tissue from 31 patients with AIDS and 12 HIV-1-negative controls was investigated. Design: Most laboratories have access to the methods used. We readily applied in situ hybridization and immunohistochemistry to archival formalin-fixed paraffin-embedded (FFPE) brain specimens. Methods: The techniques used to detect HIV-1 were explant culture, in situ hybridization with 35-labeled polymerase (pol) gene riboprobes and immunohistochemistry with monoclonal antibody to gp41. Results: HIV-1 was isolated from explant cultures in 13 out of 20 (65%) patients, whereas HIV-1-infected cells were detected in FFPE brain tissue from nine out of 26 (35%) patients examined by in situ hybridization and in seven out of 26 (27%) patients examined by immunohistochemistry. Conclusions: Although the isolation technique was the most sensitive of the three techniques tested, infected cells may be identified with in situ hybridization in conjunction with immunohistochemistry.