Β© 2015, American Society for Microbiology. Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (Ξ²-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the Ξ²-Co. Vs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several Ξ±-Co. Vs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human Ξ²-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptorbinding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of Ξ±-Co. Vs and Ξ²-Co. Vs in groups B and C bind to their specific receptor proteins. Thus, two Ξ²-Co. Vs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.