Chloroplast thylakoids contain four classes of lipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (cpPG). We have investigated the effects of these lipids on the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC), by substitution of different fractions of EPC in the membranes by the various chloroplast lipids. Damage to liposomes after freezing to −18°C was measured as carboxyfluorescein leakage or fusion between vesicles. The presence of all chloroplast lipids increased leakage. However, the maximum amount of leakage and the concentration dependence were dramatically different between the different lipids. Only SQDG induced vesicle fusion, while the non-bilayer lipid MGDG did not. The presence of MGDG in the membranes led to more leakage than the presence of another non-bilayer lipid, egg phosphatidylethanolamine (EPE). In EPE-containing liposomes, leakage was strongly associated with fusion. Combinations of different chloroplast lipids had an additive effect on leakage induced by freezing. Most of the leakage from galactolipid-containing vesicles occurred during the first 15 min of freezing at −18°C. After a 3 h incubation period, most leakage occurred between 0°C and −10°C. Lowering the temperature to −22°C had only a small additional effect. Incubation of liposomes at −10°C in the presence of 2.5 M NaCl without ice crystallization, approximately the same concentration obtained by freezing to −10°C, resulted in very little leakage. Air drying of liposomes to low water contents resulted in massive leakage, both from pure EPC vesicles and from vesicles containing galactolipids. The latter vesicles showed more leakage at any given water content than EPC vesicles.