Coronavirus spike (S) glycoproteins mediate receptor binding， membrane fusion， and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor， whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs 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 β-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 receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain， where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus， two β-CoVs in group A， HKU1 and murine CoV， have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.