A cationic bilayer adsorbed to a self-assembled monolayer (SAM) of alkylthiols could be a useful substrate for DNA immobilization and patterning. Therefore, we studied the layer formation of a self-assembled system consisting of a base layer of a negatively charged SAM chemisorbed on gold, a middle layer of an electrostatically adsorbed cationic bilayer, and a top layer of double-stranded DNA that electrostatically adsorbs to the cationic bilayer. The formation of DNA, lipid, and alkylthiol layers was monitored by surface plasmon spectroscopy. Cationic lipids readily formed layers with thickness between 32 and 33 A on self-assembled alkylthiols possessing terminal carboxylic acid groups within 24 h and at pH > 2. Fluorescence bleaching experiments indicated that these layers were homogeneous and relatively immobile. For comparison, we found that cationic lipids do not form layers on alkylthiols possessing a terminal alcohol group, while zwitterionic lipids formed bilayers on these surfaces and on the carboxylated surfaces with a thickness of approximately 38-44 Angstrom. The use of self-assembled alkylthiols with diethylene glycol groups prohibited the formation of both cationic and zwitterionic lipid layers and also prevented DNA adsorption. Finally, DNA was adsorbed to cationic lipid bilayers which were electrostatically attached to negatively charged SAMs. The results indicate that DNA forms a layer of 8 Angstrom calculated thickness, which is consistent with a monolayer possessing average interhelical distances of 50 Angstrom, in agreement with other studies using different techniques. Hence this surface is useful for immobilizing DNA. No differences were observed in kinetics of deposition or the thickness of the DNA monolayer when different cationic lipids were used.