TMPRSS2 activates the SARS-CoV spike (S) protein for membrane fusion and reduces viral control by the humoral immune response. The S protein of SARS-CoV is proteolytically activated by cathepsins B and L upon viral entry into endosomes. However, the role of host cell proteases in the secretory pathway or on the surface of target cells in cleaving SARS S is unclear. Previous studies showed that TMPRSS2 activates influenza virus hemagglutinin and human metapneumovirus F protein. Here, it is shown that TMPRSS2 cleaves SARS S. Western blot analysis revealed that SARS S is cleaved into fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). Cis-cleavage results in release of SARS S fragments into the supernatant and inhibition of antibody-mediated neutralization, likely due to SARS S fragments acting as antibody decoys. Trans-cleavage activates SARS S on effector cells for fusion with target cells and allows efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. ACE2 and TMPRSS2 are coexpressed by type II pneumocytes, important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, TMPRSS2 may promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion. SARS-CoV causes SARS, which claimed 800 lives in 2002-2003. SARS-CoV-related viruses were identified in bats and palm civets, and it is believed that human contact with these animals introduced SARS-CoV into the human population. The S protein mediates entry into target cells by engaging ACE2. Changes in the S protein sequence reflect adaptation to efficient usage of the human receptor, likely a prerequisite for high viral pathogenicity. The SARS S protein is an important determinant of viral cell and species tropism. The SARS S protein comprises 1,255 amino acids and contains 23 N-linked glycosylation sites. It is synthesized in the secretory pathway and incorporated into the viral envelope and plasma membrane. The S protein has a domain organization of class I fusion proteins, containing an N-terminal surface unit (S1) and a C-terminal transmembrane unit (S2). Proteolytic activation by host cell enzymes is a key feature of many class I fusion proteins. A study showed that proteolytic activation of SARS S is mediated by cathepsins in target cells, most importantly by cathepsin L. The efficiency and biological relevance of STMPRSS2 activates the SARS-CoV spike (S) protein for membrane fusion and reduces viral control by the humoral immune response. The S protein of SARS-CoV is proteolytically activated by cathepsins B and L upon viral entry into endosomes. However, the role of host cell proteases in the secretory pathway or on the surface of target cells in cleaving SARS S is unclear. Previous studies showed that TMPRSS2 activates influenza virus hemagglutinin and human metapneumovirus F protein. Here, it is shown that TMPRSS2 cleaves SARS S. Western blot analysis revealed that SARS S is cleaved into fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). Cis-cleavage results in release of SARS S fragments into the supernatant and inhibition of antibody-mediated neutralization, likely due to SARS S fragments acting as antibody decoys. Trans-cleavage activates SARS S on effector cells for fusion with target cells and allows efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. ACE2 and TMPRSS2 are coexpressed by type II pneumocytes, important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, TMPRSS2 may promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion. SARS-CoV causes SARS, which claimed 800 lives in 2002-2003. SARS-CoV-related viruses were identified in bats and palm civets, and it is believed that human contact with these animals introduced SARS-CoV into the human population. The S protein mediates entry into target cells by engaging ACE2. Changes in the S protein sequence reflect adaptation to efficient usage of the human receptor, likely a prerequisite for high viral pathogenicity. The SARS S protein is an important determinant of viral cell and species tropism. The SARS S protein comprises 1,255 amino acids and contains 23 N-linked glycosylation sites. It is synthesized in the secretory pathway and incorporated into the viral envelope and plasma membrane. The S protein has a domain organization of class I fusion proteins, containing an N-terminal surface unit (S1) and a C-terminal transmembrane unit (S2). Proteolytic activation by host cell enzymes is a key feature of many class I fusion proteins. A study showed that proteolytic activation of SARS S is mediated by cathepsins in target cells, most importantly by cathepsin L. The efficiency and biological relevance of S