Alkylphosphocholines are a new class of anticancer agents also representing a potential source for the treatment of tropical diseases caused by Leismania and Trypanosoma parasites. Most of the reported data suggest that mitochondria might be a main target, although direct mitochondrial effects of alkylphosphocholines have never been studied so far. In this work we show that erucylphosphohomocholine, a new antineoplastic alkylphosphocholine, permeabilized the inner membrane of isolated rat liver mitochondria, causing (i) a decrease of the inner membrane potential, (ii) an increase in state 4 respiration, (iii) a significant decrease in the rate of ATP synthesis, and (iv) low amplitude swelling of mitochondria at concentrations of 25–50 uM. In the presence of cyclosporin A, it strongly inhibited mitochondrial respiration. At 100 uM, erucylphosphohomocholine caused biphasic high amplitude swelling of mitochondria. On the other hand, oligomycin or cyclosporin A partially protected certain human cancer cell lines against apoptosis induced by this drug. Our findings demonstrate that direct mitochondrial alterations might be involved in anticancer and antiprotozoal activities of alkylphosphocholines. (Supported by DIME, National University of Colombia, Medellin Branch; by a grant from the Volkswagen‐Stiftung, Germany, VWZN2047; and Genzyme Pharmaceuticals (Liestal, Switzerland).