To explore the role of the transmembrane domain of the HN protein in the structure and function of the molecule, three conserved leucine residues in this domain which occur in a heptad-repeat motif were changed to alanine singly or in combination by site-specific mutagenesis. None of the mutant proteins were defective in translocation and intracellular transport. All mutant proteins formed disulfide-linked dimers. However, tetrameric structures of proteins with mutations in the third or most carboxy-terminal leucine could not be detected by sucrose gradient analysis, and mutant proteins with changes in both the first and second leucine formed less-stable tetramers. These results suggest that the transmembrane domain plays a role in the tetrameric structure of the HN protein. These mutations also altered the biological activities of the protein. Mutant proteins with alterations in the third leucine were very defective in attachment activity and somewhat defective for neuraminidase activity while all other mutant proteins had wild-type levels of attachment and neuraminidase activity. While all mutant proteins showed diminished fusion-promotion activity, proteins with mutations in the third leucine and proteins with changes in both the first and second leucines were very defective in fusion promotion. These results suggest that elimination or destabilization of the tetrameric structure affects attachment activity and fusion-promotion activity of the HN protein.