The nucleotide sequence of gene 5 encoding the rotavirus nonstructural protein NSP1 (NS53) of 6 strains (EW, EHP, RRV, I321, OSU, and Gottfried) was determined and compared to 6 previously reported strains (SA11, UK, RF, Hu803, DS-1, and Wa). The 12 rotavirus strains were derived from a total of five separate species (murine, bovine, simian, porcine, and human). Gene sizes ranged from 1564 to 1611 nucleotides in length and the deduced protein sequences were found to be 486 to 495 amino acids in length. Comparisons of NSP1 amino acid sequences showed identities ranging from 36 to 92%. This diversity was most evident between strains from different species. Phylogenetic analysis revealed a clustering of NSP1 sequences according to species origin with the exception that the human and porcine strains were included in a single grouping. Northern blot hybridizations using additional rotavirus strains from the five species confirmed the grouping found by sequence analysis. The species specificity of NSP1 is consistent with the hypothesis that NSP1 plays a role in host range restriction.