Langmuir monolayers have been extensively investigated by various experimental techniques. These studies allowed an in-depth understanding of the molecular conformation in the layer, phase transitions, and the structure of the multilayer. As the monolayer is compressed and the surface pressure is increased beyond a critical value, usually occurring in the minimal closely packed molecular area, the monolayer fractures and/or folds, forming multilayers in a process referred to as collapse. Various mechanisms for monolayer collapse and the resulting reorganization of the film have been proposed, and only a few studies have demonstrated the formation of a bilayer after collapse and with the use of a Ca2+ solution. In this work, Langmuir isotherms coupled with imaging ellipsometry and polarization modulation infrared reflection absorption spectroscopy were recorded to investigate the air-water interface properties of Langmuir films of anionic nucleolipids. We report for these new molecules the formation of a quasi-hexagonal packing of bilayer domains at a low compression rate, a singular behavior for lipids at the air-water interface that has not yet been documented.