Avian H5N1 influenza A viruses are considered to be of high pandemic potential as they are able to cross the avian-human species barrier and cause disease in humans. In the present study we assessed the impact of amino acid substitutions in the hemagglutinin (HA) of antigenic escape mutants of influenza A/Mallard/Pennsylvania/10218/84 (H5N2) (Mld/PA/84-MA) virus on the level of neutralizing antibodies and the ability to protect mice against challenge with the wild type H5 influenza virus. beta-Propiolactone-inactivated vaccines prepared from eight different H5 escape mutants could be separated into two groups based on levels of protection. One group of escape mutants [m46(7), m46(7)-24B9, m46(7)-55, and m46(7)-55-24B9] was characterized by providing high levels of protection (90.0-95.4% survival) to mice against subsequent challenge with 5 LD(50) of wild type Mld/PA/84-MA virus. The other group of escape mutants [m176/26, m55(2), m55(2)-24B9, and m24B9-176/26] provided moderate level of protection (57.1-66.6% survival) in mice. Analysis of the amino acid substitutions in the HA revealed that two amino acid changes in antigenic site B of the HA molecule (D(126)-->N and K(152)-->N) were associated for decreases in the levels of antibody and the immune protection afforded by vaccination with these H5 virus escape mutants. The phenotypic effects of mutations in HA gene of H5 virus may be of importance to appraise the extent and direction of H5 influenza viruses antigenic evolution.