The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel ฮฒ-sheets, ฮฒ5 and ฮฒ6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire ฮฒ6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the ฮฒ6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the ฮฒ6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the ฮฒ6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the ฮฒ6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein. ยฉ 2006 Elsevier Inc.