A two-fold increase in the amount of phospholipids was observed in Babesia divergens infected human red blood cells. In vitro incubation with [32P]-phosphorus and [3H]-glycerol demonstrated that B divergens has the ability to synthesize the phospholipid backbone. On the other hand, the low incorporation of [14C]acetate indicated the absence of a de novo fatty acid synthesis and suggested the necessity of an exogenous lipid source for the parasite. Several intra-erythrocytic growth cycles of B divergens could be achieved in vitro, using a serum-free medium supplemented only with fractions of human high density lipoproteins (HDL). At an HDL concentration of 0.5 mg/ml (protein concentration) and with a 1% starting parasitaemia, parasite growth was similar to that observed under standard culture conditions with 10% human serum, at least for the first 24 h, a time equivalent to three parasite erythrocytic life-cycles. Lipid transfer from HDL to the intra-erythrocytic parasites was demonstrated by uptake and exchange of fluorescent NBD-phosphatidylcholine (NBD-PC) loaded HDL at different temperatures. Kinetic experiments with [3H]-oleyl-PC-loaded HDL demonstrated a unidirectional transfer of lipids from radiolabelled HDL to the parasite; partial conversion of PC to phosphatidylethanolamine (PE) was also observed. In the semi-defined medium, the HDL fraction appeared to be the major source of lipids for the growth of B divergens in human erythrocytes.