Two cell lines, CNS-5 and CNS-6, were established by cocultivation of sedimented cells in cerebrospinal fluid (CSF) from two anti-human T-lymphotropic virus type I (HTLV-I) antibody-positive male patients with encephalopathy and HTLV-I-associated myelopathy, respectively, with peripheral blood mononuclear cells from a healthy seronegative female. These cell lines, possessing a normal female karyotype, revealed similar characteristics as follows; they expressed HTLV-I-related antigens, they produced C-type retrovirus particles, HTLV-I provirus genomes were integrated into their DNAs, and they had CD4+ activated T-cell markers. In addition, immunocytochemical and immunoelectron microscopic studies showed peculiar immunoreactivity of these cell lines with anti-alpha/beta T-cell antigen receptor (TCR) antibodies; beta Fl, defining beta chain epitope, was only positive in the perinuclear spaces and rough endoplasmic reticulum in some cells, and WT31, recognizing alpha/beta framework, was mostly negative, while CD3 was expressed in the majority of the cells. These facts indicate that HTLV-I-infected cells were present in CSF of these two patients, and suggest that neurological disorders associated with HTLV-I may not be restricted to myelopathy and may include brain abnormalities.