Inspired by advances in cell surface display technology [22, 23] and peptide fragment library construction [24] , we devised a highthroughput scheme that utilizes yeast display and FACS that allows for direct screening of random viral peptide libraries against complex antisera instead of isolated antibodies (Zuo T, Shi XL, Xu WH, Lin Z, and Zhang LQ, unpublished results). The peptide library is generated by random digestion of the gene encoding the target viral protein, followed by a PCR-based reassembly step that results in fragments with controllable lengths (normally in the range of 100-500 bp) [24] . These peptides are then expressed on the yeast cell surface by fusion to the yeast adhesion receptor AGA 2 protein, which has previously been used to display defined short antigen fragments of various viral and non-viral proteins [19] [20] [21] . From the sequences of the antigenic peptides after screening, it is possible to determine the relative frequency of each amino acid residue involved in recognition by antisera, and how these residues are distributed in the three-dimensional structure of HA. Using this approach, we analyzed the 2009 A(H1N1) influenza virus HA protein using mouse, goat and human antisera. Unexpectedly, different antigenic profiles were seen for different antisera. Moreover, five antigenic regions were identified, four of which are located in the conserved HA stem region responsible for membrane fusion. ELISA binding assays and absorption experiments using peptides that encompass these regions have confirmed their antigenic activities.