Mixed self-assembled monolayers provide an attractive model system for investigating the role of different molecules in biological membranes. This paper describes the preparation and characterization of a novel type of mixed monolayer assemblies composed of thiol analogues of cholesterol and fatty acid. The mixed : monolayers are prepared by coadsorbing 11-mercaptoundecanoic acid (MUA) and thiocholesterol(cholest-5-ene-3 beta-thiol, TC) from solution directly onto evaporated gold surfaces. The influence of TC on the molecular composition and conformation in the mixed monolayer is analyzed by using a combination of infrared reflection-absorption spectroscopy (IRAS), X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurement, and cyclic voltammetry. The results indicate that the TC molecules maintain their conformation in the mixed monolayers, whereas the MUA molecules display a significantly more disordered conformation as compared to the MUA molecules in the pure monolayer. Cyclic voltammetry shows that the mixed monolayers are more densely packed and less permeable than the pure TC and MUA monolayers. The kinetics of the coadsorption of TC and MUA from ethanol indicates that adsorption of TC initially is strongly preferred over MUA but that MUA dominates over TC at long coadsorption times. This is because there is a larger energy gain per unit area in forming monolayers with MUA, Further, it is also seen that the number of molecules per unit area changes with the molecular composition, as a consequence of the different sizes of TC and MUA. We present herein a method for calculating the mole fraction of TC on the gold surface, (chi TC), which accounts for this variation. As a consequence of the dissimilar size and shape of the two molecules, the wetting properties of the mixed monolayer are found to be mainly governed by the fractional area of TC, rather than by the molecular composition of TC, (chi TC).