Monoclonal antibodies specific for the hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus were used to examine the antigenic structure of HN and its role in the initiation of infection and immunity. Using 10 anti-HN antibodies, four distinct antigenic sites designated I-IV were topographically mapped on the HN molecule by competitive-binding assays. To relate the biological functions of HN to its antigenic structure, anti-HN antibodies were analyzed for their inhibitory activity in neuraminidase, hemagglutination, and hemolysis inhibition tests. Antibodies to antigenic site I inhibited hemagglutination and one of these antibodies also inhibited neuraminidase activity. Antibodies to site II inhibited neither activity. However, hemolysis an F protein activity was inhibited, suggesting that these antibodies which bind to HN interfere with F-mediated fusion. Antigenic sites III and IV had different effects on the hemagglutinating and neuraminidase functions of HN: Site III antibodies inhibited hemagglutination while antibodies to site IV only inhibited neuraminidase activity. Antibodies to each antigenic site inhibited virus production. Since antibodies to sites I and III inhibited hemagglutination, it is likely that they block virus adsorption. Antibodies to HN site II only inhibited hemolysis, and therefore, may prevent virus penetration. Antibodies reacting with site IV inhibited virus production after virus penetration. Since neuraminidase activity was the only function inhibited, the viral enzyme may be involved in virus release. The fact that site IV antibodies inhibited neuraminidase but not hemagglutination suggests that these sites are distinct.