Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.