We have synthesized a series of pyrene/cholesterol co-functionalized adlayers on quartz, oxidized silicon, indium-doped tin oxide and gold substrates. The pyrene derivative is N-1-pyrenesulfonyl-ethylenediamine (PSEDA) and the cholesterol derivative is cholesterol-ethylenediamine (Chol-NH2), which was bound covalently to substrates through epoxide functionalities. X-ray photoelectron spectroscopy shows covalent attachment of both moieties. Optical ellipsometry shows an increase of ca. 5 angstrom with pyrene/cholesterol co-attachment on oxidized silicon wafers, and an increase of ca. 12 angstrom when only pyrene was added. Steady-state fluorescence measurements indicate the presence of cholesterol reduces the efficiency of pyrene excimer formation and provides a less polar environment as sensed by the PSEDA I-1/I-3 band ratio. The amount of pyrene excimer formed depends oil the reaction little for the adlayer co-deposition reaction. Cyclic voltammetry shows that covalently bound PSEDA is oxidized at ca. 540 mV and physisorbed PSEDA is oxidized at, ca. 780 mV. AC voltammetry shows that Chol-NH2 in the adlayer reduces the electron transfer rate for the PSEDA redox reaction. (c) 2007 Elsevier Ltd. All rights reserved.