Surface-enhanced Raman scattering (SERS) spectra of four amphiphilic nucleolipids in single-layer Langmuir-Blodgett (LB) films deposited on silver island film substrates from pure water and complementary nucleotide-containing subphase and corresponding powder normal Raman spectra were obtained. The analysis of these spectra indicates that the SERS effect is mainly caused by a charge-transfer mechanism, and only the nucleobase headgroup moieties and complementary bases combined with them through hydrogen bonds, which are directly in contact with the silver island film substrates, could be enhanced. For the amphiphilic nucleolipids with the identical nucleobase headgroups, the SERS spectra of the LB films are similar, implying that the orientations of these nucleobase moieties on the silver substrates are analogous. However, the nucleobase takes different orientations on the silver substrates before and after complementary binding. The nucleobases in the LB films deposited from pure water are nearly lying flat on the silver surface, while the complementary binding pairs transferred from the air/water interface tend to take an end-on orientation on the metal surface. (C) 2003 Elsevier Inc. All rights reserved.