The surface conformation and accessibility of oligonucleotides within arrays are two key parameters that affect the utility of immobilized nucleic acids in sensor technologies. In this work, a novel combination of analytical techniques was used to compare two methods for DNA immobilization on glass. The aim of the study was to identify a method that generated a high surface density of hybridization-accessible oligonucleotides in a true planar monolayer. The first method based on direct coupling of silanized DNA to the glass surface showed a high immobilization density of 0.013 molecules/nm2 but low surface accessibility, as shown by the hybridization measurements (< or =15%). The second method, based on the biotin-streptavidin interaction, generated a high immobilization density (0.02 molecules/nm2) and high surface accessibility (90%). Atomic force microscopy and X-ray photoelectron spectroscopy indicated that both methods achieved uniform surfaces. Using the biotin-streptavidin system, the intermolecular distance between the hybridized molecules could be tightly controlled by titrating biotinylated complementary and noncomplementary oligonucleotides.