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The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero.sup.1-3. Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells.sup.4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients. Expression of TMPRSS2--a protease that activates SARS-CoV-2 for entry into cells--renders SARS-CoV-2 insensitive to chloroquine., Author(s): Markus Hoffmann [sup.1] [sup.2] , Kirstin Mösbauer [sup.3] [sup.4] , Heike Hofmann-Winkler [sup.1] , Artur Kaul [sup.1] , Hannah Kleine-Weber [sup.1] [sup.2] , Nadine Krüger [sup.1] , Nils C. [...]