We have characterized thiol-derivatized, single-stranded DNA (5'-HS-(CH2)(6)-CAC GAC GTT GTA AAA CGA CGG CCA G-3', abbreviated HS-ssDNA) attached to gold via a sulfur-gold linkage using X-ray photoelectron spectroscopy (XPS), ellipsometry, and P-32-radiolabeling experiments. We found that hybridization of surface-bound HS-ssDNA is dependent on surface coverage. The buffer concentration of the HS-ssDNA solution was found to have a profound effect on surface coverage, with adsorption greatly reduced at low salt concentration. More precise control over surface coverage was achieved by creating mixed monolayers of the thiol-derivatized probe and a spacer thiol, mercaptohexanol (MCH), by way of a two-step method, where first the gold substrate is exposed to a micromolar solution of HS-ssDNA, followed by exposure to a millimolar solution of MCH. A primary advantage of using this two-step process to form HS-ssDNA/MCH mixed monolayers is that nonspecifically adsorbed DNA is largely removed from the surface. Thus, the majority of surface-bound probes are accessible for specific hybridization with complementary oligonucleotides and are able to discriminate between complementary and noncomplementary target molecules. Moreover, the probe-modified surfaces were found to be stable, and hybridization reactions were found to be completely reversible and specific in a series of experiments where duplex melting was examined.