To utilise liposomes for permeation measurements, the temperature dependence of permeation rate had to be established first. A lipid bilayer can exist in the gel phase or in the liquid disordered phase, which differ dramatically in their permeation properties. The phase transition temperature of the three-component lipid bilayer with cholesterol, which was used in this work, has been previously shown to be 41.5 °C. In a permeation assay, the liposomes should not be permeable at laboratory temperature, but it should be possible to start permeation by raising temperature. Three temperatures were investigated: 30 °C, 40 °C, and 50 °C. The experiment was run for 15 minutes. The time dependence of the relative amount of CF released (Fig. ) reveals that at 30 °C, which is safely below the phase transition temperature, there was no permeation throughout the measurement period. At the other extreme at 50 °C, which is well above the phase transition temperature, permeation was too rapid, and it would be inaccurate to evaluate permeability from only a few data points. A suitable temperature thus proved to be 40 °C, which was just below the phase transition but close enough for CF permeation to already occur at a reasonable rate. The measured CF release curve (time dependence of concentration over time taken from the inflexion point onwards) was regressed by an algebraic model, detailed in the Methods section. An excellent agreement between the model and experiment was obtained (Fig. ). The liposomal permeability of CF in PBS medium had a value of (1.4 ± 0.4) ‧ 10 -8 cm/s, which is consistent with previously reported values obtained from the COSMOPerm calculation (≈10 -8 cm/s) . Furthermore, the partition coefficient was evaluated for this sample according to Eq. 3.2, which had a value of 1.6 ± 0.1. This value is again consistent with COSMOPerm calculation (≈1).