Lymphocytes from the central nervous system (CNS) of animals with acute experimental autoimmune encephalomyelitis (EAE) have been isolated and characterized. The lymphocytes were separated from Lewis rats which were injected either with an emulsion of myelin basic protein (BP) emulsified in complete Freund's adjuvant (CFA) to cause EAE or with CFA alone as a control. Using density gradient centrifugation, from 9 days post-inoculation (d.p.i.) (before clinical signs appear), to 19 d.p.i. (after signs abate), lymphocytes were recovered from the spinal cords and popliteal lymph nodes of BP-injected animals. Lymphocyte cell number, phenotype, and antigen specificity were determined. Results show that the onset of clinical signs correlated with lymphocyte influx into the CNS. A clinical index of 1 was associated with less than 10(6) cells per gram of CNS wet weight (cells/g CNS) while animals with a clinical index of 4 had more than 15 X 10(6) cells/g CNS. During remission, when only minor residual neurologic signs were evident, significant numbers of lymphocytes (greater than 10(7) cells/g CNS) could still be isolated. In contrast, no lymphocytes were obtained from control CNS tissue. The phenotype of the recovered cells was predominantly of the helper/inducer T cell subset (greater than 40%). Although the percentages of these cells in the CNS were increased when compared to the lymph nodes, I-A expression on CNS-isolated lymphocytes showed the most significant increase with disease progression. Recovered lymphocytes responded to both BP and CFA-related antigens indicating that both CNS-specific and CNS-non-specific inflammatory cells were present in the exudate.