Human metapneumovirus (HMPV) is a major causative agent of severe bronchiolitis and pneumonia. Its fusion (F) protein must be cleaved by host proteases to cause membrane fusion, a critical step for virus infection. By generating Vero cells constitutively expressing the transmembrane serine protease TMPRSS2 and green fluorescent protein-expressing recombinant HMPV, we show that TMPRSS2, which is expressed in the human lung epithelium, cleaves the HMPV F protein efficiently and supports HMPV multiplication. The results indicate that TMPRSS2 is a possible candidate protease involved in the development of lower respiratory tract illness in HMPV-infected patients. Copyright © 2008, American Society for Microbiology. All Rights Reserved.