Persistent strains of Theiler's virus, a murine picornavirus, produce a life-long infection of the central nervous system of the mouse and induce a chronic demyelinating disease. Strain DA1, a molecular clone of such a persistent strain, produces a prominent cytopathic effect in BHK-21 cells but is less efficient at infecting L929 cells. We cloned the cDNA of a derivative of virus DA1, adapted to promote a rapid cytopathic effect in L929 cells. Adaptation of the new variant (named KJ6) to L929 cells correlated with an enhanced viral entry rather than with an increased replication rate of the genome. Mutations responsible for L929 cells adaptation occurred in amino acids exposed at the surface of the capsid, in the CD loop of VP1 (100-102) and in the EF loop of VP2 (162-171-173), suggesting that these residues could be involved in receptor recognition. These two clusters of amino acids are precisely known to be part of neutralization epitopes. They also differentiate persistent from neurovirulent strains of Theiler's virus. Adaptation of the virus to L929 cells was accompanied by attenuation of its virulence for the mouse. Taken together, these data suggest a close relationship between receptor binding, virus neutralization, and virus phenotype.