Two influenza virus substrains, A/RI/5-/57 and A/RI/5+/57, with antigenically similar hemagglutinins and neuraminidases but with different properties of elution from erythrocytes, have been examined for the specificity of their interaction with cell surface sialyloligosaccharides. This was accomplished by using erythrocytes treated with Vibrio cholerae neuraminidase to remove sialic acids, and then modified with CMP-NeuAc and three purified sialyltransferases to contain either the NeuAc alpha 2,3Gal, NeuAc alpha 2,6Gal, or NeuAc alpha 2,6GalNAc linkages on cell-surface glycoproteins. Each virus was tested for its ability to adsorb to and hydrolyze sialic acid from the derivatized cells. The hemagglutinins of the A/RI/5-/57 and A/RI/5+/57 viruses were found to have totally different specificities, binding respectively to the NeuAc alpha 2,3Gal and NeuAc alpha 2,6Gal linkages as preferred receptor determinants. In contrast, the neuraminidases of the two viruses exhibited similar specificities, efficiently hydrolyzing only the NeuAc alpha 2,3Gal linkage. The results suggest that the difference in the ability of the A/RI/5-/57 and A/RI/5+/57 viruses to elute from erythrocytes resides in the different receptor specificities of their hemagglutinins. Indeed, it appears that A/RI/5-/57 virus elutes because its receptor determinant, NeuAc alpha 2,3Gal, is rapidly hydrolyzed by the viral neuraminidase, while the A/RI/5+/57 virus fails to elute because its preferred receptor determinant NeuAc alpha 2,6Gal is largely resistant to hydrolysis.