Two temperature sensitive mutants, ts3 and ts11, of the WSN (HON1) strain of influenza virus, belonging to the same recombination group, have a 1000- to 10,000-fold lower infectivity titer and lack hemagglutinating and neuraminidase activity when grown at nonpermissive temperature (39.5°), compared with virus grown at permissive temperature (33°). The patterns of viral polypeptides synthesized in cells infected with these mutants are similar to those found with wild type virus. Neuraminidase activity of the mutant viruses is more temperature labile than the enzyme of the wild type. Despite the low yield, morphologically intact virus particles are found at 39.5°, but in contrast to virus grown at 33° large aggregates of virus accumulate near the cell surface. These aggregated virus particles contain neuraminic acid as demonstrated by a colloidal iron stain. Thus, it is likely that a neuraminic acid containing protein serves as receptor for the hemagglutinin of other virus particles, resulting in the extensive aggregation. Hemagglutinating activity of virus grown at 39.5° is restored by treatment of the aggregates with neuraminidase from Vibrio cholerae. These results suggest that the lack of hemagglutinating activity of mutant virus grown at 39.5° is a consequence of the formation of aggregates of virus particles carrying neuraminic acid on their surface, and that the ts defect is in the neuraminidase but not the hemagglutinin molecule. It is postulated that neuraminidase is essential for the replication of influenza viruses and is required to remove neuraminic acid from the viral envelope to avoid aggregation of the progeny virus.