Genetic studies have demonstrated that susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is multigenic with linkage to the MHC class I locus, H-2D. We have analyzed the effect of mutations (H-2bm13 and H-2bm14) in the H-2Db gene on central nervous system (CNS) virus replication, virus-specific delayed type hypersensitivity (DTH) and disease induction in mutant [bm14D2F1 and bm13D2F1] and parental B6D2F1 hybrids. The results indicate that substitutions of only a single residue (bm14D2F1) or three residues (bm13D2F1) in H-2D in the mutant leads to a sequence of events culminating in disease susceptibility. Mutation of the H-2D gene is associated with reduced or delayed virus clearance following the acute phase of exponential CNS virus growth and an increased level of virus persistence. Concomittant with the greater virus antigen burden, mutant mice respond with higher levels of virus-specific DTH and develop inflammatory demyelinating lesions.