Monolayer behavior of the dye 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled analogues of phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) were studied by a variety of methods. Attachment of the NBD chromophore onto either the phospholipid headgroup or the aliphatic chain significantly changes the parent phospholipid monolayer properties. In contrast to that of the condensed-type DPPE monolayer, the isotherm of the DPPE-NBD derivative with NBD in the headgroup exhibits a liquid-expanded/liquid-condensed phase transition plateau while the isotherm of the acyl-chain-labeled NBD(C-12)-PC shows liquid-expanded behavior lacking the plateau observed in the isotherm of DPPC. Surface potential and spectroscopic data revealed that the NBD group on both DPPE-NBD and NBD(C-12)-PC embeds into the phospholipid headgroup region of the monolayer, thus suggesting a looping of the NBD-labeled chain of NBD(C-12)-PC toward water. Polarized fluorescence and red-edge excitation shift studies revealed a regular alignment of uniformly oriented NBD chromophores in the DPPE-NBD monolayer controlled by networks of intermolecular hydrogen bonds. By contrast, the NBD group on NBD(C-12)-PC experienced a less-structured environment permitting rotation of the NBD chromophore and fast relaxation of surrounding water dipoles. Organization of the NBD-labeled phospholipids at the air/water interface and intermolecular interactions responsible for the monolayer structuring are discussed.