Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion5. In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein6. Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment (Supplementary Fig. S2). To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes (EEs) or endolysosomes (ELs) was analyzed by immunofluorescence analysis (IFA) and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions (anti-NP, red) were observed in early endosome antigen 1 (EEA1)-positive EEs (green), while more virions (34.3%) were transported into the late endosomal–lysosomal protein LAMP1+ ELs (green) (n > 30 cells for each group). By contrast, in the presence of CQ or HCQ, significantly more virions (35.3% for CQ and 29.2% for HCQ; P < 0.001) were detected in the EEs, while only very few virions (2.4% for CQ and 0.03% for HCQ; P < 0.001) were found to be co-localized with LAMP1+ ELs (n > 30 cells) (Fig. 1b, c). This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV7.