Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active antileishmanial drug. Transient HePC treatment of Leishmania donovani promastigotes at 10 μM significantly reduced the phosphatidylcholine content and enhanced the phosphatidylethanolamine (PE) content in parasite membranes, suggesting a partial inactivation of PE- N -methyltransferase. Phospholipase D activity did not seem to be affected by HePC. In addition, the enhancement of the lysophosphatidylcholine content could be ascribed to phospholipase A2 activation. Moreover, transient HePC treatment had no effect on the fatty acid alkyl chain length or the fatty acid unsaturation rate. Concerning sterols, we found a strong reduction of the C ₂₄ alkylated sterol content, and the enhancement of the cholesterol content could be the result of the HePC condensation effect with sterols. Because some of the effects observed after transient HePC treatment were different from those previously observed in HePC-resistant parasites, it could be hypothesized that continuous in vitro drug pressure induces the mechanisms of regulation in Leishmania lipid metabolism.