Molecular recognition in Langmuir monolayers at the air-water interface as a function of headgroup orientation and substrate using isotherms and in-situ Fourier transform infrared (FT-IR) reflection spectroscopy has been investigated. Isotherm measurements show that urea and 2,4,6-triaminopyrimidine (TAP) are specifically bound to barbituric acid lipid monolayers. As expected, TAP causes a larger shift. in the limiting area of the isotherms than urea due to steric requirements. The peak positions of the CH stretching vibrations of the barbituric acid lipids indicate that the alkyl chains of barbituric acid lipids 1-3 are in a close-packed all-trans conformation both before and after the recognition process. The complexation of TAP is monitored in situ using external reflection FT-IR spectroscopy through shifts of the conformation-sensitive carbonyl frequencies in the head group region, while no shifts in the carbonyl peaks are observed when urea recognizes the lipid monolayers.