More precise understanding of the immune response against T-cell receptors (TCRs) is a prerequisite for successful TCR vaccination therapy of multiple sclerosis and other neurological autoimmune diseases. We conducted a detailed analysis of a paradigmatic anti-TCR response, using synthetic TCR peptides and highly purified recombinant TCR V alpha and Vbeta variable chains for the selection of CD4+ T-cell lines from a healthy volunteer. The target TCR (designated TCR(HWBP-3)) was obtained from HWBP-3, an autologous CD4+ T-cell line specific for myelin basic protein. The V alpha and Vbeta chains of TCR(HWBP-3) were expressed in Escherichia coli and purified by Ni-chelate chromatography and SDS (sodium dodecyl sulphate) gel electrophoresis. Further, we synthesized a set of 13- to 22-mer peptides spanning the complementarity-determining regions (CDR) 1, 2 and 3 and the framework regions (FR) of the alpha and beta chains of TCR(HWBP-3). The TCR peptides and proteins were then used to select a panel of TCR-specific CD4+ T-cell lines from donor HW. Several T-cell lines cross-reacted with a recombinant V chain and synthetic peptide. Cross-reactive immunogenic TCR epitopes were identified in the FR1 and CDR3 regions of the TCR(HWBP-3) alpha chain and in the FR1, CDR1 and CDR2 regions of the TCR(HWBP-3) beta chain. The TCR proteins and peptides were recognized in the context of at least three different HLA-DR molecules [DR2a (DRB5*0101), DR2b (DRB1*1501) and DRB1*1401/DRB3*0202]. Notably, the majority of the TCR peptide-selected T-cell lines did not react with the full-length recombinant V chains, suggesting they recognize 'cryptic' determinants. Based on the diversity of the anti-TCR immune response, we suggest that candidate TCR peptides should be screened in vitro in functional experiments before they are clinically applied for TCR vaccination therapy.