The aim of the present study was to evaluate the toxicity and the activity of a new lipid complex formulation of amphotericin B (AMB) (LC-AMB; dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and AMB) that can be produced by a simple process. Like other lipid formulations, this new complex reduced both the hemolytic activity of AMB (the concentration causing 50% hemolysis of human erythrocytes, >100 microg/ml) and its toxicity toward murine peritoneal macrophages (50% inhibitory concentration, >100 microg/ml at 24 h). The in vivo toxicity of the new formulation (50% lethal dose, >200 mg/kg of body weight for CD1 mice) was similar to those of other commercial lipid formulations of AMB. The complex was the most effective formulation against the DD8 strain of Leishmania donovani. It was unable to reverse the resistance of an AMB-resistant L. donovani strain. In vivo LC-AMB was less efficient than AmBisome against L. donovani.