A method was further developed to screen non-tissue-culture-adapted bovine rotaviruses for serotype, using a neutralization test with infectious fecal rotavirus. One of those rotaviruses (B223) which was not blocked by antiserum to the neonatal calf diarrhea virus (NCDV) serotype was then adapted to cell culture in the presence of the antiserum for two or more passages and hyperimmune antiserum to this isolate had a 60-fold-higher homologous neutralization titer than with the NCDV serotype rotavirus. Seventy-three isolates were serotyped and eight (11%) were not of the NCDV serotype (bovine rotavirus serotype I). Of these eight, five belonged to the new bovine rotavirus serotype II and three were not typed, indicating the existence of one or more further serotypes. Cross-protection studies in gnotobiotic calves showed that cross-protection only occurred between rotaviruses of the same serotype, and even a minor serotype difference was sufficient for the calves to show a lack of cross-protection. The serotypes (I and II and the three untyped isolates) also showed differences in the rate of migration in polyacrylamide gel electrophoresis of some of their RNA segments (no. 4, 6, 7, 8, 9, 10), indicating that they were of different electropherotypes.