B10.RIII mice develop chronic and relapsing experimental autoimmune encephalomyelitis (EAE) after immunization with the myelin basic protein (MBP) peptide 89–101. The disease is associated with the major histocompatibility complex (MHC) (eae1). We have now investigated the importance of non–MHC regions for the EAE susceptibility in a cross between RIIIS/J and B10.RIII mice which share the MHC region but differ in disease susceptibility. Linkage analysis using microsatellite markers spanning the genome identified a region (eae2) on chromosome 15 which showed linkage to disease (P=0.0002). Our data also suggest linkage to a second region (eae3) on chromosome 3 (P=0.0024), and provide evidence for locus interactions between eae2 and eae3. These results provide clues to the genetic basis of multiple sclerosis.