Temperature dependence of the free energy barrier for formation of (A) I1 state (), (B) I2 state (), and (C) pore formation () for control vesicles (open circles) and vesicles containing 3 mol% hexadecane (solid circle). Measurements were carried out in 10 mM MES, 100 mM NaCl, 1mM CaCl2, 1 mM EDTA, pH 5.0 at a total lipid concentration of 0.2 mM. Units of free energy are arbitrary and represent Kcal/mol if the unknown constant A = 1 sec−1. Curves through the data were obtained using the equation , with the parameters and their variances given in Table S3.