The DNA-directed self-assembly of surface-bound layers of gold nanoparticles offers a broad range of applications in biomedical analyses as well as in materials science. We here describe a new concept for the assembly of substrate-bound nanoparticle monolayers which employs bifunctional nanoparticles as building blocks, containing two independently addressable DNA oligomer sequences. One of the sequences was utilized for attaching the particle at the solid support, while the other sequence was used to establish cross-links between adjacently immobilized particles. AFM analyses proved the functionality of inter-particle cross-links leading to enhanced surface coverages and the formation of monolayered supramolecular aggregates attached to the substrate. We anticipate that further refinement of this approach will enable applications, for instance, the assembly of ordered layers useful as transducers in biosensing. (C) 2003 Elsevier Inc. All rights reserved.