The JHM strain of mouse hepatitis virus (JHMV) establishes a persistent infection in the murine central nervous system (CNS) associated with chronic ongoing demyelination in the absence of detectable virus. To distinguish between immune and replication associated mechanisms of persistence, brains from acutely and persistently infected mice were analyzed for viral RNA mutations in the encapsidation sequence (ECS) and regions encoding either the transmembrane domains of the matrix (M) protein or a protective CTL epitope in the nucleocapsid (N) protein. Detection of the ECS to 120 days post infection (p.i.) indicated low levels of replication. The ECS remained stable whereas the fragment encoding the CTL epitope revealed extensive diversity with mutation frequencies in the order of 2.0 per 1000 nts. The M gene also remained stable despite random mutations during the acute phase. Mutations in the N gene were random and not selected for during persistence, with the exception of a single prominent Pro363 to Ser substitution in a region not associated with any known regulatory function or immune response. Mutations within the CTL epitope affecting CTL recognition were found early in responder BALB/c mice (H-2d), but also in non-responder C57BL/6 (H-2b) mice, suggesting that CTL escape variants play no significant role in establishing persistence.