Rabbit antibody, F2C12, was prepared against a synthetic peptide of 13 amino acids corresponding to the C-terminus of the F2 subunit of Sendai virus F protein. F2C12 was shown to bind specifically to the F2 subunit, irrespective of proteolytic cleavage of the F protein. F2C12 did not affect the infectivity, hemolytic, and cell fusion activities of activated virus, but F2C12 did inhibit proteolytic cleavage of the precursor F0 to subunits, F1 and F2, by trypsin, factor Xa from chick embryos and Tryptase Clara from rat lungs. When F2C12 was added to the chorioallantoic cavity of chick embryos, cleavage activation of Sendai virus was inhibited. Intranasal administration of F2C12 to Sendai virus-infected rats suppressed activation and multiplication of progeny virus in the lungs. These results indicate that proteolytic cleavage of the F protein, with a single arginine residue at the cleavage site, occurs extracellularly in the allantoic cavity of chick embryos and in the bronchial lumen of rats.