The interactions between sex hormones (estradiol and testosterone) and a 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine (DMPC) molecular layer were investigated by observing the liposome and the surface area (A) vs. surface pressure (π) of the DMPC monolayer at an air–water interface. The shape and size of the liposome changed with the addition of estradiol and testosterone. The π–A isotherm, the time variation of the surface pressure, and the microscopic observation of monolayer were examined to clarify the different effects of the steroid hormones. These experimental results are discussed in relation to the formation of a rigid membrane due to hydrogen bonding between estradiol and DMPC.