The mixed monolayer behavior of dipalmitoylphosphatidylcholine (DPPC) and dihexadecyl phosphate (DHDP), two of the major components in the preparation of liposomes, on both water and phosphate buffer subphases was investigated from the measurements of surface pressure-area per molecule (II-A) isotherms. The II-A isotherms indicated that the two components were miscible at the air/water interface. The miscibility and nonideality of the mixed monolayers were examined by calculating the excess area as a function of composition, and deviations from ideality were observed, which suggests that the existence of attractive interactions between DPPC and DHDP molecules in the mixed monolayers on a water subphase. Nevertheless, the buffer subphase environment enhanced the dissociation of DHDP, and the presence of ions in the subphase may also disturb molecular interactions and packing in the mixed monolayers, resulting in more expanded monolayers and complicated excess area behavior. Furthermore, the excess free energies of mixing and free energies of mixing were evaluated from the isotherms, and the most stable state of the mixed monolayers on a water subphase was found with X-DHDP = 0.5 or 0.6 However, the tendency for DPPC and DHDP to form a mixed monolayer was less significant on the phosphate buffer subphase than on a water subphase.