The role of the cytoplasmic domain of the Newcastle disease virus fusion protein in syncytia formation was explored by characterizing the intracellular processing and activities of proteins with deletions and point mutations in this region. Deletion of the entire domain (amino acids 523 to 553) resulted in a protein which was minimally proteolytically cleaved and had no syncytia forming activity. Deletion of the carboxy terminal half of the domain (amino acids 540 to 553) resulted in a protein that was normally processed but had no syncytia forming activity. Deletion of amino acids 547 to 553 resulted in a protein with approximately 30% wild-type levels of activity while deletion of amino acids 550 to 553 yielded a protein with wild-type activity. The results suggested that amino acids 540 to 550 are important for syncytia formation and this conclusion was supported by two internal deletions as well as point mutations in this region. Mutation of two cysteine residues in and adjacent to the transmembrane domain, which are potential sites for fatty acid acylation, had no effect on syncytia formation either singly or in combination.