AbstractWe here assessed thein vitroefficacy of the naptho-quinone buparvaquone (BPQ) againstBesnoitia besnoititachyzoitesin vitro. BPQ is currently licensed for the treatment of theileriosis in cattle in many countries, but not in the EU. In 4-day treatment assays, BPQ massively impaired tachyzoite proliferation with an IC50of 10 ± 3 nm, and virtually complete inhibition was obtained in the presence of nmBPQ. Exposure to 1µmBPQ leads to ultrastructural changes affecting initially the mitochondrial matrix and the cristae. After 96 h, most parasites were largely distorted, filled with cytoplasmic amylopectin granules and vacuoles containing components of unknown composition. Host cell mitochondria did not appear to be notably affected by the drug. However, upon prolonged exposure (14–16 days) to increased BPQ concentrations,B. besnoititachyzoites exhibited the capacity to adapt, and they resumed proliferation at dosages of up to 10µmBPQ, albeit at a lower rate. These BPQ-adapted parasites maintained this lower susceptibility to BPQ treatment after freeze–thawing, and inspection by the transmission electron microscopy revealed that they underwent proliferation in the absence of structurally intact mitochondria.