In this report we study the interaction of reovirus type 3 Dearing (RV3) with vertebrate erythrocytes whose membrane glycoconjugates differ in the degree and position of O-acetylation of their sialic acid (NeuAc) residues. Binding to erythrocytes required the presence of NeuAc on cellular glycoconjugates, since pretreatment with sialidase (neuraminidase) abolished hemagglutination by RV3. Furthermore, we found that RV3 binds efficiently to and hemagglutinates all erythrocyte preparations possessing exclusively NeuAc, or a mixture of NeuAc and 4-O-acetyl-NeuAc (4-O-Ac-NeuAc), but poorly to erythrocytes bearing a mixture of 9-O-Ac-NeuAc and NeuAc, suggesting that RV3 binds preferentially to NeuAc-containing glycoconjugates. To gain further evidence for this hypothesis we treated chicken erythrocytes with influenza C virus neuraminate, 9-O-acetylesterase, to convert their 9-O-Ac-NeuAc residues to NeuAc. When hemagglutination assays were carried out on these cells, we observed a 16-fold increase in the hemagglutination titer for RV3 compared to untreated cells. When we treated bovine submaxillary mucin (BSM) with influenza C virus, we observed a dramatic increase in its potency as an inhibitor of RV3 hemagglutination. Concomitant with this, the 9-O-Ac-NeuAc residues on BSM were converted to NeuAc. Taken together and in conjunction with a previous report (A. F. Pacitti and J. R. Gentsch, 1987, J. Virol. 61, 1407–1415), these results suggest that the virion attachment protein exhibits a strong preference for NeuAc over 9-O-Ac-NeuAc as a receptor component on erythrocytes.