Multiple sclerosis (MS) is a putative autoimmune disease that is linked with HLA-DR2,w15. Proteolipid protein (PLP) is a candidate autoantigen in MS, but the disease-associated epitopes have not been determined. Using overlapping and non-overlapping PLP peptides, we have studied the T cell response to the major hydrophilic domain PLP 85-159 in the peripheral blood of MS and healthy subjects (HS). Short-term T cell lines (TCL) were selected against each peptide using microwell plates and the frequency of peptide-specific TCL was estimated. PLP 95-116-specific TCL were most efficiently generated and the frequency was significantly higher in MS compared with HS (P < 0.05). When compared between DR2,w15+ and DR2,w15- MS, TCL frequency to PLP 95-116 was significantly higher in DR2,w15+ MS (P < 0.005) and TCL reactive to the overlapping peptide 105-124 were also increased in DR2,w15+ MS (P < 0.025). Using DR gene-transfected L cells, we could show that the DRB1*1501 product of the DR2 haplotype presents PLP 95-116 to TCL selected against the peptide. These results imply that PLP 95-116 represents a major epitope for the DR2,w15+ MS.