The physical origin of the extremely high thermal stability of tetraether liposomes composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been investigated. The leakage rate of trapped 5, 6-carboxyfluorescein (5(6)CF) and the proton permeability in PLFE liposomes have been measured using fluorescence probe techniques in the temperature range of 25-85 degrees C. The results are compared with those obtained from nonarchaebacterial liposomes. Egg yolk phosphatidylglycerol (eggPG) and PLFE liposomes exhibit similar large negative zeta-potentials (-31 to -34 mV) and low permeability coefficients for 5(6)CF, indicating that membrane surface charge is responsible for the low leakage rate of 5(6)CF in PLFE liposomes. This assertion is confirmed by the observation of an increased leakage rate of 5(6)CF with decreasing membrane surface negative charge via varying the content of egg yolk phosphatidylcholine (eggPC) in eggPC/eggPG binary mixtures. Gel-state dipalmitoylphosphatidylcholine bilayers and PLFE liposomes exhibit similar permeability coefficients for 5(6)CF, suggesting that lipid packing also plays an important role in the low leakage rate of 5(6)CF. PLFE liposomes, especially those approximately 60 nm in diameter, are remarkably thermally stable in regard to proton permeability, which increases by less than 2 x 10(-10) cm/s from 25 to 82 degrees C. The proton permeability comparison of various liposomes reveals that the tight and rigid lipid packing is the major contributor of the extremely low proton permeation in PLFE liposomes; the inositol moiety and the branched methyl groups may also contribute, but to a much lesser extent.