Leishmania donovani promastigote lines resistant to hexadecylphosphocholine (HePC, miltefosine) at 2.5, 5.0, 10.0, 20.0 and 40.0 microM were developed in vitro by continuous step-wise drug pressure. The 40 microM line was 15 times more resistant to HePC than the wild-type clone and showed cross-resistance to the ether lipid ET-18-OCH3 (edelfosine) but not to the standard anti-leishmanial drugs. Resistance was stable up to 12 weeks in drug-free culture medium. No amplification of specific genes, including the multidrug resistance P-glycoprotein gene, could be detected in the resistant parasites.