To locate the various functions associated with the hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus in the primary structure of the protein, a temperature-sensitive (ts) mutant and seven antigenic mutants were sequenced. The ts mutant was defective in its ability to agglutinate erythrocytes and infect host cells, while its neuraminidase activity was normal. Its sequence revealed two closely spaced amino acid substitutions (residues 262 and 264) and one distant substitution (residue 461). Revertants could not be isolated, suggesting that more than one of the substitutions is responsible for the defective hemagglutinating activity. The antigenic mutants were selected with monoclonal antibodies that delineate four nonoverlapping antigenic sites (I-IV) and separately inhibit hemagglutinating, neuraminidase, and hemolysis activities. Mutants selected with antibodies to antigenic sites I-III were used to map these functions on the primary sequence of HN. Each antigenic mutant had a single point mutation in the HN gene that resulted in an amino acid substitution in the protein. A site II mutant selected with an antibody which inhibits hemolysin activity had a substitution at amino acid 420, while a mutant selected with antibody that inhibits only erythrocyte binding (site III) had a substitution at amino acid 541. Two antigenic mutants selected with an antibody that inhibits hemagglutination and neuraminidase activities (site I) had amino acid substitutions in close proximity (residues 277 and 279) to the two closely spaced substitutions of the ts mutant. These findings suggest that the region defined by the ts mutant and these two antigenic mutants is involved in host cell binding. Antigenic mutants selected with another site I antibody had amino acid changes at residue 184, indicating that antigenic site I is discontinuous in the primary sequence. This antibody blocks only hemagglutination, but mutants selected with it had a decreased neuraminidase activity. This finding supports the idea that the neuraminidase site is close to, but distinct from, the hemagglutination site.