Atomic force microscopy (AFM) was used to study oligonucleotide adsorption on a gold surface. Oligonucleotides are thiolated at the 5 end with SH(CH2)6-5 to adsorb them specifically via a gold−sulfur linkage. Thanks to nanoscopic resolution of AFM, we have been able to measure the density of adsorbed molecules, varying experimental parameters. The adsorption kinetics of the oligonucleotides shows a two-step mechanism. The first step is a nonspecific adsorption of molecules. In the second one, these oligonucleotides diffuse and react with gold. This reaction depends on the structure of the surface (steps and/or reconstructions). The rate, which increases with temperature, leads to an activation energy of thiol dissociation on gold around 100 kJ/mol.