A total of 16 different strains of rotavirus derived from seven mammalian species (four each from human and porcine species, two each from equine and simian species, and one each from canine and bovine species) and two avian species (one each from turkeys and chickens) were examined in plaque-reduction neutralization tests. Seven anti-genically distinct serotypes were established on the basis of a ≥20-fold difference between titers of homologous and heterologous reciprocal neutralizing antibodies. Serotypes 1 (strain Wa) and 2 (strain DS-I) were recovered only from humans. Serotype 3 included human rotavirus strain WALK 57/14, rhesus monkey rotavirus strain MMUI8006, vervet monkey rotavirus strain SA-ll, dog rotavirus strain CU-I, and horse rotavirus strain H-2. The newly established serotype 4 was identified in both humans (strain St. Thomas no. 4) and pigs (strains Gottfried, SB-IA, and SB-2). Porcine (strain OSU) and equine (strain H-I) rotaviruses made up a possible fifth serotype. Bovine rotavirus (strain NCDV) constituted a sixth serotype, and chicken rotavirus (strain Ch 2), which had a prime-strain relation with turkey rotavirus (strain Ty 1), was designated serotype 7. A surprising observation that emerged from this study was the existence of a rotavirus (porcine strain SB-IA) bridging serotypes 4 and 5.