2. Influenza virus degraded by freezing and thawing, by treatment with 5 M urea, or by heating at 65°C. still combined with homologous antibody and was demonstrable by blocking of the hemagglutination-inhibition and virus neutralization reactions.

3. After 50 cycles of freezing and thawing, much of the blocking antigen activity was not sedimented by centrifugation at 120,000 g for 2 hours, and electron microscopy showed complete disruption of the virus particles. So called soluble blocking antigen was obtained from four strains of influenza A, the Lee strain of influenza B, mumps, and Newcastle disease viruses.

4. Soluble blocking antigens from influenza A viruses were highly strain-specific; gave little or no reaction in complement-fixation tests; stimulated but little antibody production in rabbits and did not induce immunity in mice; caused reactivation of infective virus in neutral mixtures of homologous virus and immune serum.

5. Repeatedly frozen and thawed influenza virus preparations did not interfere with the propagation of infective virus in the allantoic sac. The blocking antigen activity they contained was precipitated by half saturated ammonium sulfate, destroyed by trypsin, chymotrypsin, or heating at 56°C. for 30 minutes, but was unaffected by desoxyribonuclease or ribonuclease.

6. These findings are in accord with the view that soluble blocking antigen obtained from influenza virus particles on disruption by repeated freezing and thawing is protein in nature and represents the essential antigenic material of the intact virus.