Monolayers of a cytosine-based nucleolipid (1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) (ammonium salt), CDP-DG) at basic subphase have been prepared at the air-water interface both in absence and presence of guanine. The formation of the complementary base pairing is demonstrated by combining surface experimental techniques, i.e., surface pressure (pi)-area (A), Brewster angle microscopy (BAM), infrared spectroscopy (PM-IRRAS) and computer simulations. A folding of the cytosine-based nucleolipid molecules forming monolayer at the air-water interface occurs during the guanine recognition as absorbate host and is kept during several compression-expansion processes under set experimental conditions. The specificity between nitrogenous bases has been also registered. Finally, mixed monolayers of CDP-DG and a phospholipid (1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt), DMPA) has been studied and a molecular segregation of the DMPA molecules has been inferred by the additivity rule. (C) 2018 Elsevier Inc. All rights reserved.