The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69-89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule. Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide. Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system. The recombinant molecule consists of the Valpha2 domain connected to the Vbeta8.2 domain via a 15-residue linker. Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure. C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69-89) peptide, as analyzed using surface plasmon resonance. Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness.