Primary cultures from a brain biopsy specimen of a human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) seropositive patient with progressive dementia contained small numbers of monocytoid cells and showed reverse transcriptase activity that persisted for as long as 100 days. Electron microscopy of these cells revealed the presence of HTLV-III/LAV virions. Subcultured cells removed from primary cultures by trypsinization were nonspecific esterase negative and did not express virus or show evidence of HTLV-III/LAV proviral sequences, while those remaining in the original flasks were nonspecific esterase positive and continued to produce virus. Virus from primary cultures was transmitted to peripheral blood-derived monocyte-macrophages and T cells. Virus production in T-cell cultures was transient while the monocyte-macrophages, like the primary cultures, produced virus for at least 120 days. Infection of several brain-derived cells with this and another HTLV-III/LAV isolate failed to demonstrate virus replication. These results indicate that the HTLV-III/LAV-infected cells recovered from the brain of this patient are cells of the mononuclear phagocyte series.