Investigations of central nervous system cellular immune reactivity in human disease, as reflected in the responses of cerebrospinal fluid lymphocytes, have been limited primarily due to the low numbers of cerebrospinal fluid lymphocytes available during routine diagnostic lumbar punctures in normal individuals and most patients with demyelinating diseases. We report the use of a T-cell growth factor generated by by phytochemagglutinin-stimulated, irradiated normal peripheral blood lymphocytes to maintain long-term proliferating cultures of human cerebrospinal fluid lymphocytes. Cerebrospinal fluid lymphocytes. Cerebrospinal fluid T-cell cultures were initiated from 10 to 14 cerebrospinal fluid samples with up to 5000-fold expansion of initial cell numbers. Few, if any, macrophage or surface immunoglobulin-bearing cells were present, while 80 to 90% of the cultured cells were T cells as demonstrated by rosette formation with sheep red blood cells. Mixed lymphocyte cultures with cultured cerebrospinal fluid T cells and irradiated, freshly isolated allogeneic peripheral blood lymphocytes yielded a positive response in four of the five cultures tested.