Some children infected by HIV-1 demonstrate nervous system disease. Because a significant percentage of these children are believed to be infected during gestation and it is thought that HIV-1 may infect distinct glial populations, this work tested the hypothesis that different HIV-1 isolates can infect cells of the developing human fetal central nervous system (CNS). Central nervous system organotypic tissue cultures derived from human fetal brain enable the study of complex interactions between CNS cell types. Central nervous system organotypic cultures were exposed to lymphocytotropic (L-tropic) or monocytotropic (M-tropic) HIV-1 isolates and monitored for viral infection. HIV-1 gp41 and p24 antigens were detected by immunocytochemistry (ICC), HIV-1 RNA was localized in the cytoplasm of CNS cells by in situ hybridization (ISH), and viral DNA was detected by polymerase chain reaction (PCR) in HIV-1-exposed cultures. Double-label ICC identified HIV-1 antigens in both microglia and astrocytes. These results demonstrate that both L- and M-tropic isolates infect microglia and astrocytes in human fetal organotypic cultures. In addition, HIV-1 infection was detected in culture supernatants up to day 57 postinfection and at 90 days by coculture with susceptible CEM cells. HIV-1 infection of neural cells appears to be productive. This model may permit further examination of the interaction of HIV-1 with the developing human CNS and the mechanisms of AIDS-associated neuropathology.