Alternative methods to the standard hemagglutination inhibition (HI) and neutralization tests to probe the antigenic properties of the influenza virus hemagglutinin (HA) were developed in this study. Vaccinia virus recombinants expressing reference HAs were used to immunize rabbits from which polyclonal antibodies were obtained. These antibodies were subtype specific but showed limited intra-subtype strain specificity in ELISA. The discriminatory capacity of these antibodies was however markedly increased after adsorption to cells infected with heterologous influenza viruses, revealing antigenic differences that were otherwise undistinguishable by standard HI and neutralization tests. Furthermore, the unadsorbed antibodies could be used to select escape mutants of the reference strain which after sequencing unveiled amino acid changes responsible of the noted antigenic differences. These procedures therefore provide alternative methods for the antigenic characterization of influenza HA and might be useful in studies of HA antigenic evolution