Although human T-lymphotropic virus type I (HTLV-I) has been implicated in the etiology of tropical spastic paraparesis (TSP) and HTLV-I associated myelopathy (HAM), the direct infectivity of the virus against constituent cells in the central nervous system remains undetermined. To investigate the neurotropism of HTLV-I, we exposed cultured human and simian glial cells to HTLV-I. Primary mixed glial cell cultures of astrocytes and oligodendrocytes were obtained from adult human and cynomolgus monkey (Macaca fascicularis) brains by an enzyme digestion-Percoll gradient method. After two weeks in vitro, the cells were co-cultured with irradiated MT-2 cells, an HTLV-I-producing T-cell line. Cultures were double stained with antibodies against cell-type specific markers and anti-HTLV-I p19 (gag) monoclonal antibody. The HTLV-I antigen was demonstrated in small numbers of glial fibrillary acidic protein-positive cells (astrocytes) and galactocerebroside-positive cells (oligodendrocytes) in both the human and simian cultures. Electron microscopy demonstrated the presence of type C virus-like particles in the cytoplasm of astrocytes. These results indicate that HTLV-I is capable of infecting human and simian glial cells in vitro.