Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease 1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-19 4-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans. Infection with SARS-CoV-2 is characterized by initial mild disease associated with respiratory symptoms at the peak of viral replication 1,8. In some patients, a late severe immunological syndrome occurs 6-14 days after the onset of symptoms that may require intensive care and is responsible for most of the fatalities 1-3. HCQ has well-documented in vitro activity against various viruses 4 and has emerged as an active compound against SARS-CoV-2 in different screening programmes, including a library of 1,520 Food and Drug Administration (FDA)-approved compounds 5. In Vero E6 cells, HCQ has a 50% maximal effective concentration (EC 50) 5,9,10 that varies between 0.7 and 4 μM. It may inhibit viral transport in endosomes by alkalinizing the intra-organelle compartment 10,11 and affect glycosylation, as reported for other viruses 12. The drug may also act as an immunomodulatory agent 13,14. In patients with lupus, HCQ decreases the level of inflammatory cytokines 11,15,16 , which may be relevant for the treatment of COVID-19 2. Furthermore, it has been proposed that AZTH, which displays in vitro antiviral activity against SARS-COV-2 5,17 , could potentiate the efficacy of HCQ 6. On the basis of these properties, HCQ has been considered for the treatment of COVID-19, alone or in combination with AZTH 6,7. We and others have set up non-human primate (NHP) models of SARS-CoV-2 infection 18-20. Here we used cynomolgus macaques (Macaca fascicularis) to test different treatment strategies with HCQ, alone or in combination with AZTH, before or after the peak of viral replication. We also tested HCQ administration as pre-exposure prophylaxis treatment...