Neutralization assays on calf fecal rotavirus with antisera to two previously described bovine rotavirus serotypes allowed the isolation of four rotaviruses belonging to a distinct third serotype. In a survey of 85 calf isolates, 80 rotaviruses belonged to serotype 1 (91%), 1 belonged to serotype 2 (1%), and 4 belonged to serotype 3 (5%). Serotypes 1 and 2 were shown to not cross-protect in a passive immunization experiment in gnotobiotic lambs. Ingestion of specific antiserum protected against infection with the homologous, but not heterologous, serotype. Rabbits with no previous exposure to rotavirus responded to sequential vaccination with bovine and human rotavirus serotypes with antibody specific to those serotypes, and the response did not broaden to include serotypes to which they had not been exposed. These factors suggested the need for multivalent rotavirus vaccines. By contrast, 47 adult cows on 11 farms had neutralizing antibodies to two bovine and three human rotavirus serotypes. After vaccination with one bovine rotavirus serotype, these cows produced a significant increase in antibody titers to these same five serotypes but not to two other serotypes to which they had no preexisting antibody. These results were interpreted to indicate that cows will respond heterotypically after monovalent vaccination to all rotavirus serotypes with which they have had experience and, therefore, that single serotype vaccination may be sufficient. This conclusion has practical importance for rotavirus immunization procedures.