Temperature-sensitive mutants of bovine rotavirus, UK Compton strain, and rhesus monkey rotavirus, MMU18006 strain, were used to derive 16 reassortants by coinfection of MA104 cells. The parental viruses differed phenotypically in their neutralization specificity, their ability to hemagglutinate, and their requirement for exogenous trypsin for infectivity. When the reassortants were assayed for neutralization specificity and hemagglutination, four phenotypes were observed, indicating that these two rotaviral functions segregated independently. Protease-enhanced infectivity phenotype segregated with the HA phenotype indicating that these two functions were manifestations of the same gene product. In order to determine the gene responsible for these rotaviral functions, the reassortants were genotyped by hybridizing 32P-labeled parental transcripts and denatured reassortant genomic RNAs and analyzing the resulting hybrids by gel electrophoresis. The fourth RNA segment was clearly shown to code for HA and protease enhanced plaque formation in MA104 cells. The neutralization antigen was linked to the eighth and ninth RNA segments that comigrated during gel electrophoresis and thus could not be differentiated.