In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimalba ctericidal concentration (MBC) of HCQ against the mobile spirochetes was >32 μg/mlat 37°C, and >128 μg/ml at 30°C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37°C, the MBC for young biologically active cysts (1-day old) was >8 μg/ml, but it was >32 μg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ was ≥MBC. At high concentrations of HCQ (256 lg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ was ≥MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was ≥MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.