The liposomal assay employed in this work allows direct observation of permeation enhancement in a single layer of phospholipids. Two well-known permeation enhancers with different enhancement mechanism were studied: ethanol and oleic acid. Permeation enhancement was investigated at 30 °C, as no CF release occurred at this temperature under normal conditions. The effect of ethanol was investigated by stepwise addition of small quantities of ethanol (40 µl in each step) to a spectrophotometric cuvette containing a sample of liposomes containing CF. A stepwise release of CF from the liposomes was observed (Fig. ) after the addition of each ethanol aliquot. Ethanol is known to cause lipid extraction from the membrane and to subsequently form a second phase together with the extracted lipids . After each ethanol addition, release from the affected liposomes was immediate but other liposomes remained intact as the added ethanol was bound with the extracted lipids. The increment of CF release in each step corresponds to the liposomes whose membrane integrity was disrupted by ethanol addition.