To define the sequence changes that occurred in an avian influenza virus neuraminidase (NA) during the evolution of virulence, we have studied the NA of the virulent and avirulent A/Chick/Penn/83 (H5N2) influenza viruses. A comparison of the deduced amino acid sequence from these viruses shows that the virulent strain, which evolved from the avirulent by the accumulation of point mutations (Bean et al., 1985), acquired four amino acid changes in the NA: one in the transmembrane segment, one in the stalk, and two in the head. A comparison of the deduced amino acid sequences with those of the human N2 NAs indicates a 20-amino acid deletion in the stalk of the Chick/Penn/83 NA. Antigenic analysis of the NAs from the avirulent and virulent Chick/Penn/83 virus shows they are antigenically very closely related, but can be distinguished with two monoclonal antibodies at a site which probably involves at least one of the amino acid changes in the NA head. Antigenic analysis also shows the Chick/Penn/83 NAs are closely related to the NAs of other N2 avian influenza viruses isolated between 1965 and 1984, supporting previous studies which indicate a relative antigenic stability of the NA among avian N2 influenza viruses. The Chick/Penn/83 NAs are the first N2 NA genes of an avian virus to be sequenced. These NAs are antigenically closely related to the 1957 human N2 NAs, and show a high degree of amino acid sequence homology with the prototype 1957 human N2 NA. These data give further support to the view that the 1957 human H2N2 viruses were at least partially derived from an avian source.