Surface properties of Sendai virus envelope membrane have been measured, using both biological and biophysical techniques. Both normal and trypsin-treated virus were studied. SDS gel electrophoresis showed cleavage of the F protein exclusively by trypsin. The major activity change was observed in the hemolysing activity which is an expression of F protein. Hemolysis was reduced to less than 10% of its value for intact virus. 31P nuclear magnetic resonance studies of the envelope surface of the native virus showed a highly restricted phospholipid headgroup environment. Interestingly, this restriction was relieved by treatment with trypsin. Thus these data suggest a role of the F protein of Sendai virus in tightly organizing the surface of the viral envelope membrane.