We have developed a measurement method for the cytochrome P450 3A4 (CYP3A4)-mediated oxidation of cholesterol in lipid membranes. This method is based on the sterolstructural selectivity for the nanopore formation of streptolysin O (SLO) in a lipid membrane. A decrease in the fraction of 4 beta-hydroxycholesterol in lipid membranes decreased the nanopore forming ability of SLO. The incubation of the liposomes consisting of L-alpha-phosphatidylcholine (PC), L-alpha-phosphatidylethanolamine (PE), and cholesterol (0.98:0.02:0.75, mol/mol) with CYP3A4 decreased the nanopore forming ability of SLO, indicating the conversion of cholesterol to 4 beta-hydroxycholesterol. The change in nanopore forming ability did not occur in the presence of erythromycin, which is an inhibitor of CYP3A4.