The long-chain lipid derivatives of acyclovir-a nucleoside analogue were used to prepare Langmuir-Blodgett (LB) films, including the single-chained derivative (SGSA) and the double-chained derivative (DASA). The bilayer LB film of DASA or the SGSA/cholesterol (SGSA/Chol) mixture (1:1, mol/mol) on quartz plates was investigated with ultraviolet absorption spectroscopy, and the blue-shifted absorption with 4 nm (DASA) or 18 nm (SGSA/Chol) wavelength changes was observed in comparison with their solutions in chloroform. The rigid double chains of DASA prevented adjacent molecules from approach, while the flexible single chains of SGSA did not. Then the strength of intermolecular hydrogen bonding between the nucleoside moieties of DASA was much more weaker than one of SGSA, and their blue-shifted wavelength in LB films was different. DASA and SGSA/Chol also showed the different bilayer LB films on mica according to the atomic force microscopic observation. The former was prone to tilting on solid supports while the latter would like to stand vertically with the help of cholesterol that could insert into the flexible single chains of SGSA. The chain number (one or two) and state (flexible or rigid) of lipid derivatives of nucleosides strongly impact intermolecular hydrogen bonding and self-assembly behavior. (c) 2005 Elsevier B.V. All rights reserved.