We have investigated the morphology of dimyristoyl phosphatidyl choline (DMPC)-cholesterol mixed monolayer formed on the water surface by dropping method using surface tension measurement (STm), Brewster angle microscopy (BAM), and infrared external reflection spectroscopy (IERS). STm results showed negative deviation of the limiting molecular area (A 0 ) of cholesterol occurred when cholesterol was added to the DMPC monolayer. BAM images showed the expandable DMPC monolayer changed to the condensed rigid monolayer at more than cholesterol mole fraction (x Chol ) 0.4. IERS recordings showed that the addition of cholesterol at x Chol = 0.4 occurred structural change from gaucheto transconformation of two DMPC molecule alkyl chains. From these results, it is found that cholesterol molecule has specific properties that cause structural transition of DMPC molecule alkyl chains.