The genetic changes occurring in the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) during persistent infection in the mouse central nervous system (CNS) were studied. RNase T1-oligonucleotide fingerprinting of the RNAs of 28 BeAn viruses isolated at various times postinfection (p.i.) demonstrated that mutation occurred throughout the infection. Although plaque-purified BeAn virus was used to inoculate mice intracerebrally, genetically different viruses were recovered from the CNS. One to three oligonucleotide changes were found up to Day 152 p.i., but all three viruses isolated at Day 180 had four to nine oligonucleotide changes. No pattern of oligonucleotide changes occurring in different virus isolates was found, yet three viruses isolated from different animals at Day 180 had the same four new oligonucleotides. Overall, the number of oligonucleotide changes represented a 0.1 to 1.2% change in the virus genome. In addition, the analytical two-dimensional gel technique of P.Z. O'Farrell, H.M. Goodman, and P.H. O'Farrell (Cell 12, 1133-1142, 1977) suggested that mutation occurred in all virus isolates. In nine isolates, one to three proteins were found to have charge changes, and in general, as many nonstructural proteins had charge changes as structural proteins. P20, a nonstructural protein probably equivalent to the protease described for encephalomyocarditis virus, was found to have shifted cathodally in six different viruses. Several virus isolates had doublet patterns, suggesting the possibility that within the CNS, subpopulations existed which had proteins of slightly different charge or that virus-specified proteins had been modified after translation. Finally, antigenic variation of neutralizing site(s) on BeAn virus isolates as a way for virus to evade immune surveillance and thereby maintain the persistent state was studied. The ability of mouse serum to neutralize persisting virus isolates was not significantly different from the ability to neutralize the infecting virus. Therefore, antigenic variation does not appear to be a factor in TMEV persistence.