A single-stranded oligonucleotide whose 5' end is derivatized with a mercaptohexyl tether group was either directly anchored onto a gold surface or attached to a gold surface as part of a mixed self-assembled monolayer that contains mercaptohexanol. The application of these surface-confined DNA oligomers as heterogeneous probes for the detection of polynucleotides (e.g., M13 phage DNA) is considered, with an emphasis on the elucidation of the relationship between the hybridization efficiency and the surface coverage and orientation of the probe molecules. Atomic force microscopy (AFM) and flow-injection quartz crystal microbalance (FI-QCM) were used in tandem to study the immobilization of the probe, to estimate the extent and efficiency of the hybridization of M13 phage DNA (7249 bases), and to examine the effect of using a different alkanethiol to reorient the preformed film for a higher hybridization efficiency. The surface density and the resultant hybridization efficiency were found to be highly dependent on the morphology and surface structure of the gold substrate as well as on the concentration of the solution used for the probe fabrication but much less dependent on the probe immobilization time. The lower limit of the hybridization efficiency was estimated to be about 1.1% which is an underestimate because only the resolvable circular features were included in the estimation. Although the duplex formed at the gold surface covered with only the thiolated DNA probe adopts exclusively the orientation in which the target loop is parallel with respect to the substrate surface, the predominant duplex orientation at the gold substrate modified with mixed self-assembled monolayers is tethered to the surface with a small tilt angle versus the surface normal. Visualization of the duplex orientation allows one to understand whether the Sauerbrey equation is valid for the interpretation of certain FI-QCM results. Although it is probably valid to use the Sauerbrey equation to calculate the amount of a polynucleotide at a surface covered only by the thiolated DNA probe, the practice might be questionable for that at the surface with the DNA/alkanethiol mixed SAM on the basis of our AFM images of the target orientations.