Binary monolayers comprised of mercaptopyrimidine (MP) and 1-dodecanethiol (C12SH) in varying ratios were prepared on gold surfaces by self-assembly and then characterized using reflection absorption FTIR spectroscopic (RA-IR), electrochemical, and wettability measurements. The RA-IR results revealed that the coverage of MP in the mixed monolayer is controlled by its composition in solution. The adsorption of mixed monolayers appears to be kinetically controlled. The wettability of mixed monolayers indicated that the surface fraction of C12SH exposed to the probe liquid exceeded that of its mole fraction in the monolayer. Electrochemical measurements demonstrated that the permeabilities of various quinone probes into the mixed self-assembled monolayers (SAMs) were dominated by their geometry. The origination of the geometry-dependent permeability of probes is believed to be related to the average size of MP domain in SAMs.