This paper describes a study concerning the interaction of nitric oxide with the clean Si(100)2×1 surface at room temperature. Differential reflectometry in the photon energy range of 2.4–4.4 eV, Auger electron spectroscopy (KL2,3L2,3 and L2,3VV), and low-energy electron diffraction have been used to investigate experimentally the chemisorption process of NO on the freshly cleaned Si(100)2×1 surface. Theoretically, the chemisorption process has been modeled by means of the use of quantum-chemical calculation methods [modified (intermediate) neglect of diatomic overlap, MNDO and MINDO/3]. Within this framework we have calculated the binding energy of several possible adsorption sites and the partial local density of states. The latter has been utilized to simulate the loss-corrected Si -L2,3VV Auger line shape, in the derivative mode, which proved to be a powerful tool for the interpretation of the newly revealed fine structure. The low nitrogen and oxygen coverages (15% of a monolayer) at saturation could be explained by lateral electrostatic repulsion. Also, evidence for molecular adsorption of NO on the Si(100)2×1 surface at 300 K has been found. Furthermore, the possibility that a missing-dimer defect could play a role in the adsorption process has been considered.