Arrays of Au nanoparticles were created using the inherent repeating patterns of bacterial S-layer proteins. Bacterial self-assembling S-layer protein lattices display a highly repetitive surface structure that makes them particularly suitable as biotemplates to fabricate metallic/semiconducting nanostructures and arrays. One interesting S-layer for nanoparticle templating is the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans. This S-layer, displaying hexagonal (p6) symmetry, is comprised of a hexameric protein core unit with a central pore, surrounded by six relatively large openings ("vertex points"). In this work, the influences of particle properties and adsorption conditions on the formation of ordered arrays of 5-nm Au nanoparticles using HPI S-layers were investigated. Using transmission electron microscopy (TEM), it was found that the templating of citrate-capped Au nanoparticles on HPI layers under low ionic strength conditions resulted in hexagonal-packed ordered arrays with similar to18-nm interparticle spacings that corresponded with the pore-to-pore distance of the S-layer. Interestingly, nanoparticle binding occurs at the vertex points on the HPI layer and, due to repulsion forces, adsorption tends to be favored at every second vertex point. Upon increasing the ionic strength, ordered packing is still observed. However, because interparticle repulsions are less prominent, adsorption of nanoparticles occurs in virtually every available vertex point, resulting in the formation of a honeycomb-like pattern of nanoparticles extending throughout the HPI monolayer sheet. Combined with the results of additional investigations using either uncharged hydroxy-terminated particles or positively charged ferritin molecules, the experimental data suggest that the creation of ordered arrays through biotemplating of Au nanoparticles onto HPI S-layers depends on the electrostatic interactions between individual nanoparticles as well as the interaction with the HPI layer.