A cDNA library was constructed with poly(A+) mRNA from cells infected with the virulent Italien NDV strain. A clone that hybridized to the HN gene mRNA was sequenced. A long open reading-frame encodes for a protein of 571 amino acids, with a calculated molecular weight of 61,900, including 13 cysteine residues and six potential glycosylation sites. To define the sequence changes that occurred in the avian paramyxovirus hemagglutinin-neuraminidase (HN) during the evolution of virulence, we have studied the HNs of the virulent Italien NDV strain, the mesovirulent Beaudette strain and the nonvirulent Hitchner strain. The majority of amino acid variations are conservative changes but they cluster at 4 preferential sites in the putative head of HN. The clusters of amino acid substitutions are intimately associated or overlap with regions of HN rich in charged amino acid residues and in cysteines. The latter are conserved not only between HNs from all 3 NDV strains but also between HNs of 4 different paramyxoviruses, NDV, SV 5, Sendai and PI 3. The HN coding sequence was inserted into the genome of vaccinia virus under the control of vaccinia P 7.5 K transcriptional regulatory sequences. Expression of native HN proteins at the surface of recombinant HN vaccinia-infected cells was demonstrated by indirect immunofluorescence with 2 anti-HN monoclonals.