T cell reactivity to candidate myelin autoantigens, such as myelin basic protein (MBP), may play an important role in the pathogenesis of multiple sclerosis (MS). Although MBP-reactive T cells have been found to undergo in vivo activation in patients with MS, their true precursor frequency in MS is unknown as current frequency analysis is commonly based on the T cell functional responses to MBP. In this study, we developed a TCR sequence-based ex vivo detection system using colony hybridization with oligonucleotide probes specific for CDR3 of selected T cell clones for the analysis of true T cell precursor frequency in PBMC. The results revealed that the precursor frequency of five independent T cell clones recognizing the immunodominant MBP(83-99) region was found to be in the range of 1.6 x 10(-4) in total T cells in three HLA-DR2 patients with MS compared to that of 0.25 x 10(-4) in HLA-DR2 healthy individuals. The observed frequency of MBP(83-99)-reactive T cells in MS patients was considerably higher than those measured in parallel by cell culture-based analysis (2.3 x 10(-6)) or by enzyme-linked immunospot assay (3.9 x 10(-5)) in the same peripheral blood mononuclear cell specimens. Furthermore, the study showed that MBP(83-99)-reactive T cells detected ex vivo belonged to CD45RA+, CD25+ and CD95- T cell subsets as evidenced by preferential expression of specific TCR transcripts in these cell fractions.