A mixed monolayer of 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (DHPS) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on an 1-octanethiol-modified gold substrate was visualized on the nanometer scale using in situ scanning tunneling microscopy (STM) in aqueous solution. DHPS clusters were evident as spotty domains. STM enabled us to distinguish DHPS molecules from DHPC molecules depending on their electronic structures. The signal of the DHPS domains was abolished by neutralization with Ca2+. The addition of the PS + Ca2+-binding protein of annexin V to the Ca2+-treated monolayer gave a number of spots corresponding to a single annexin V molecule.