The primary structure of the region in the outer layer protein VP3, containing the two sites associated with trypsin enhancement of infectivity of rotavirus was found to be greatly conserved in cultivable human rotavirus serotypes 1 (Wa), 2 (DS1), and 3 (P) and in four human rotaviruses directly purified from feces. Significant differences with this conserved sequence were found in human rotavirus serotype 4 (ST3), isolated from an asymptomatic neonate, and in seven animal rotaviruses. However, the two trypsin cleavage sites were conserved in every rotavirus VP3 sequence analyzed.