Virus produced in the first four days after infection of a BHK21 culture was shown to differ from that produced later in the infection. The early virus caused large plaques in IB-RS-2 cell sheets, had a slow cytopathic effect in BHK21 cultures and showed a high virulence for suckling mice. In contrast, the late virus caused small plaques, was rapid in its cytopathic effect and was of low virulence for mice. Comparison between one clone each of the early and late virus showed that no change in immunogenic specificity had taken place, but that charge changes had occurred both in VP3 and in the large trypsin-resistant fragment of VP1. The early, large plaquing clone gave rise spontaneously to small plaquing virus during the destructive phase of a single passage in BHK21 cultures. Conversely, the late, small plaquing clone gave rise to large plaquing virus after a single passage in mice. Each new virus was cloned and it was shown that they differed in VP1. This indicated that missense mutations in the genome coding for the trypsin resistant fragment of VP1 were responsible for the biological changes observed.