We have investigated a means of producing thin, oriented lipid monolayers which are stable under repeated washing and which maybe useful in biosensing or surface-coating applications. Phosphatidylcholine and the glycosphingolipid GM1 were used as representative lipids for this work. Initially, a mixed self-assembled monolayer of octanethiol and hexadecanethiol was produced on a gold surface. This hydrophobic monolayer was then brought into contact with a thin lipid film that had been assembled at the liquid/air interface of a solution, allowing the lipid to deposit on the gold surface through hydrophobic interactions. The lipid layer was then heated to cause intermingling of the fatty acid and alkanethiol chains and cooled to form a highly stable film which withstood repeated rinsing and solution exposure. Presence and stability of the film were confirmed via ellipsometry, Fourier transform infrared spectroscopy, and quartz crystal microbalance (QCM), with an average overall film thickness of similar to3.5 nm. This method was then utilized to produce GM1 layers on gold-coated QCM crystals for affinity sensing trials with cholera toxin. For these sensing elements, the lower detection limit of cholera toxin was found to be approximately 0.5 mug/mL, with a logarithmic relationship between toxin concentration and frequency response spanning over several orders of magnitude. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.