Myelin basic protein (MBP) is highly immunogenic and a known autoantigen capable of inducing experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis. We have previously described a murine monoclonal antibody (mAb), F28C4, directed against the encephalitogenic MBP peptide acetyl (Ac) 1-9, which contains a Vλx light chain. Considering the rarity of Vλx usage, we determined whether other Abs having Vλx light chains shared similar antigen (Ag) specificity. We screened a panel of Vλx-containing monoclonal and polyclonal Abs, of unknown specificity for reactivity with MBP. All such Ab, but not heavy chain isotype matched controls, bound MBP but were not polyreactive with other potential self Ags. The binding of a recombinant form of Vλx alone to MBP demonstrated the important contribution of the Vλx light chain to the reaction. With the exception of mAb F28C4 which recognizes MBP Ac1-9, the epitope specificity of all other Vλx-bearing Abs was localized to MBP residues 25-34. These results demonstrate a unique association between Vλx expression and MBP reactivity. Given that Vλx shares sequence homology with T cell receptors (TCR) from encephalitogenic T lymphocytes, these results imply a potential role for Vλx in the pathogenesis of EAE.