The hemagglutinin-neuraminidase protein (HN) of mumps virus was purified by immunoaffinity chromatography and fragmented by the combined action of CNBr and trypsin. The resulting peptides were separated by HPLC and sequenced by automated Edman degradation. Using this HN-specific amino acid sequence data, a degenerated oligo-nucleotide was produced and subsequently used to screen a mumps virus cDNA library to isolate HN-specific clones. The complete nucleotide sequence of the HN gene was determined. The monocistronic HN mRNA is approximately 1900 nucleotides long and encodes a single open reading frame of 582 amino acids. The HN protein has a unique hydrophobic stretch of 19 amino acids at its N-terminus that apparently anchors the protein in the viral envelope. A comparison of the mumps virus HN protein sequence with the sequences of the other known paramyxovirus HNs indicates that mumps virus is most closely related to SV-5, followed in decreasing order by NDV, parainfluenza virus 3, and Sendai virus. © 1988.