The formation of thiocholesterol (TC) monolayers on gold has been studied by ellipsometry, contact angle measurements, infrared spectroscopy, and cyclic voltammetry. Subsequent treatment of the TC assembly with 11-mercaptodeuterioundecanoic acid (MDUA) shows that the average surface coverage is about 65% of that of a self-assembled alkanethiolate monolayer and that it has a large number of molecular defects. These defects exist because of a mismatch between the size and shape of the TC molecule and the pinning distance at the Au(111) crystal lattice. Potential uses of these defect-rich structures are microelectrode arrays for electroanalytical and biosensor applications.