Rationale. Amiodarone interferes with the endocytic pathway, inhibits proteolysis and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles and functional consequences of amiodarone accumulation remain unclear. Objectives. To study amiodarone uptake, clarify origin of vacuoles and investigate the effect of amiodarone on the life cycle of the coronavirus responsible of the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Methods. Using alveolar macrophages, we studied uptake of ¹²⁵I-amiodarone and ¹²⁵I-B2, an analogue lacking the lateral group diethylamino--ethoxy and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. Measurements and main results. 1) amiodarone associates with different cell membranes and accumulates in acidic organelles; 2) the diethylamino--ethoxy group is an important determinant of uptake; 3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; 4) amiodarone inhibits the spreading in vitro of SARS coronavirus; 5) trypsin cleavage of the viral spike protein prior to infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. Conclusions. Amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.