The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host-range determinant for infection of mammals. Here, we demonstrate that the E to K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tract of mice and was complete by day 6 post-inoculation. This mutation correlated with efficient replication of the virus in mice.