We investigated the immune response to proteolipid protein (PLP), the most abundant central nervous system myelin protein in humans. A total of 8207 short-term T cell lines were generated from 49 individuals, 39 patients with multiple sclerosis and 10 control subjects. As we have reported previously, the frequency of PLP-reactive T cells did not differ between the two groups. To determine immunodominant PLP epitopes, proliferative responses of 971 PLP-specific lines were tested with 27 overlapping 20-amino acid peptides encompassing the human PLP sequence and the binding affinities of the PLP peptides to DRB5*0101 and DRB1*1501, DR2 MHC class II isotypes associated with multiple sclerosis, were determined. The T cell response after primary PLP stimulation was focused on two immunodominant epitopes comprising residues p30-49 and p180-199. These two fragments were recognized after processing of native protein by APCs and were situated in hydrophilic regions of PLP exhibiting only moderate affinity to DR2 molecules. In contrast, when T cells from DR2+ subjects were stimulated initially by individual synthetic peptides with either high or low affinity to DRB5*0101 and DRB1*1501 isotypes, additional cryptic epitopes were recognized. MHC restriction of lines specific for the cryptic PLP epitopes were related to binding affinity to DR2 isotypes. Our results indicate that protein Ags are recognized in vivo as immunodominant epitopes after Ag processing by APCs and as cryptic epitopes after processing, presumably by extracellular proteolytic enzymes.