A rotavirus (RV) nonstructural protein, NSP4, has recently been proposed to function as an enterotoxin in the pathogenesis of RV diarrhea. The role of NSP4 in the pathogenesis of RV diarrhea was examined by infecting cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice with virulent murine RV and by comparing deduced amino acid sequences of RV gene 10 encoding NSP4 from three distinct sets of virulent and tissue culture-adapted avirulent variant RVs. Homozygous CFTR (CFTR-/-) mice, which do not respond to any known intestinal secretagogues, experienced diarrhea comparable to that in normal CFTR+/+ littermates after RV challenge. Comparison of amino acid sequences of NSP4 from virulent and attenuated pairs of RVs failed to show consistent or significant changes. Together, these data suggest that enterotoxigenic properties of RV NSP4 are not critical in the pathogenesis of murine RV diarrhea and that attenuation of murine RVs is not usually mediated by mutations in the gene encoding NSP4.