Antigenic reactivity of a set of monoclonal antibodies (MAb) raised against the HA2 subunit of hemagglutinin of H3 subtype was characterized in a rapid culture assay. MAbs FC12 and FE1, known to recognize the same antigenic site (IV), cross-reacted with influenza viruses of H3 and H4 subtypes, regardless of their host origin. No cross-reactivity was detected with other antigenic subtypes tested (H1-H13). The involvement of conserved residues D160, N168, and F171 in the differential recognition of H3 and H4 subtypes is proposed. In contrast, MAb IIF4 that recognizes antigenic site II exhibited a broader inter-subtype reactivity including subtypes H3, H4, H5, H8 and some viruses of H2, H6 and H13 subtypes. The ability of HA2-specific antibodies to differentially react with distinct antigenic subtypes can be utilized in development of diagnostics and in the influenza virus surveillance.