We have used the mouse model to monitor the acquisition of virulence of a non-pathogenic influenza A virus upon adaptation to a new mammalian host. An avian strain, A/Mallard duck/Pennsylvania/10218/84 (H5N2) (Mld/PA/84) was adapted to mice by 23 serial lung-to-lung passages until a highly virulent mouse-adapted (MA) variant (Mld/PA/84-MA) emerged. This MA variant was characterized and compared to the parental strain as well as some of its intermediate passage variants. MA variant caused bronchopneumonia in mice with a high mortality rate (the virulence of Mld/PA/84-MA measured as log (EID50/LD50) was 1.75), while the parental, avirulent strain Mld/PA/84 did not cause illness and mortality in mice (log (EID50/LD50) was 7.25). Hemagglutination-inhibition (HAI) test with a set of hemagglutinin- (HA) specific monoclonal antibodies (MAbs) revealed antigenic differences between the parental strain and MA variant. Mld/PA/84-MA reacted with HA-specific MAbs in higher titers than the parental strain. The HA genes of the parental strain Mld/PA/84, the 1st, 3rd, 8th, and 15th intermediate passage variants, and Mld/PA/84-MA were sequenced. Three amino acid changes at positions 203, 273 and 320 were determined in the HA of MA variant. The first of them, Leu-->Pro (320), appeared in the HA stem region at the 8th passage. Two other in the HA1 globular region (Ser-->Phe (203) and Glu-->Gly (273)) appeared at the 15th passage. All of these substitutions were associated with the increase of viral infectivity for mouse lungs and changes in the HA antigenicity. The potential role of these changes in HA with respect to the process of viral interspecies transmission and acquisition of virulence for new host is discussed.