The interaction of PC-based unilamellar liposomes incorporating the complementary 5,5 di-n-dodecylbarbituric acid (DBA) or 9-hexadecyladenine (HA) in various molar ratios has been investigated using imaging, spectroscopic and calorimetric techniques. Liposomal interaction was established by turbidimetry, AFM, and optical microscopy and also by isothermal titration calorimetry. For temperatures below the main lipid phase transition, the results provide evidence for hydrogen-bonding between the adenine and the barbituric acid moieties at the lipid-water-lipid interfaces with a stoichiometry ratio of 1:1. The interaction does not lead to disruption of the liposomes but instead it affords larger aggregates. The role of cholesterol in the DBA-HA binding, at high concentrations in the PC:DBA and PC:HA bilayers has also been investigated. Evidence is found that the presence of cholesterol (at molar concentration 2:1 relative to PC) alters the DBA/HA binding mechanism. For all the systems studied, no liposomal binding could be calorimetrically detected above the main lipid phase transition temperature.