We have recently demonstrated that a strong bias for expression of V beta 8.2 is manifested early during the onset of experimental autoimmune encephalomyelitis (EAE) induced by guinea pig basic protein (Gp-BP) immunization of Lewis rats. More importantly, the V beta 8.2 bias was observed in T cells infiltrating the spinal cord (SC) and in cerebrospinal fluid (CSF), but was not present in T cells isolated from the periphery. Here, we report the V beta 8-CDR3 sequences found in unselected SC, CSF, and lymph node (LN) T cell populations at onset of Gp-BP-induced EAE. Striking similarities were observed among sequences derived from SC and CSF. Evidence for oligoclonal expansion of V beta 8.2 sequences associated with previously characterized encephalitogenic clones was observed in both SC and CSF, but not in LN. An AspSer CDR3 motif identified in encephalitogenic clones recognizing the dominant 72-89 epitope of Gp-BP was found in 9/22 SC cDNA clones, 11/24 CSF cDNA clones, and 1/16 LN cDNA clones. Interestingly, J beta 2.7 and J beta 1.3 were also highly represented in SC and CSF, but not in LN. Given that these sequences were derived from T cells present at the site of autoimmune attack and not selected by in vitro manipulation, the data offer compelling evidence that 1) selective recruitment and/or expansion of V beta 8.2+ T cells are occurring in the central nervous system; 2) these events are at least partially dependent on V beta residues which are likely to influence Ag binding; and 3) CSF-derived T cells provide a representative view of CNS events at the onset of EAE.