An enzyme inhibition assay was developed to determine methotrexate-gamma-aspartate leakage from liposomes at lipid concentrations as low as 43 nM phospholipid. When negatively charged liposomes prepared with phosphatidylglycerol/cholesterol 67:33 or phosphatidylinositol/cholesterol 67:33 were incubated in 10% (v/v) newborn calf serum, they leaked over 90% of their contents in 2 min. In contrast, liposomes prepared from phosphatidylcholine/cholesterol 67:33 leaked 18% of their contents under the same conditions. The amount of negative charge required to induce liposome leakage was determined by preparing liposomes with varying amounts of phosphatidylglycerol and phosphatidylcholine. Extensive leakage was observed only from liposomes prepared with greater than 50 mol of phosphatidylglycerol per 100 mol of phospholipid. The effect of the phase transition temperature on leakage of negatively charged liposomes in 10% (v/v) serum was investigated by using a series of phosphatidylglycerols with varying acyl chain lengths. Liposomes prepared from distearoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol leaked less than 18% of their contents in 10% serum, whereas liposomes prepared with dilauroylphosphatidylglycerol or unsaturated lipids leaked more than 70% of their contents. Lipoprotein removal from serum followed by treatment with lipid to remove residual apoproteins reduced the leakage from phosphatidylglycerol liposomes in 10% serum. Phosphatidylglycerol liposomes leaked 73% in the presence of human low-density lipoproteins, but only 29% in the presence of bovine apolipoprotein A-I, and 25% in the presence of human high-density lipoproteins. Phosphatidylglycerol/cholesterol and phosphatidylserine/cholesterol liposomes leaked 67% in 4 mg/mL bovine serum albumin purified by cold ethanol extraction. The leakage of liposomes in albumin solutions could be substantially reduced by treating the albumin with lipid.(ABSTRACT TRUNCATED AT 250 WORDS)