The formation of intermolecular complexes of two large molecules—a macrocycle and a semiaxle, which have been used in templated syntheses of amide rotaxanes—was studied by scanning tunneling microscopy (STM) and density functional theory (DFT). These experiments mimic the so-called “threading process”, which is based on intermolecular recognition and which is essential for the rotaxane synthesis in solution. First, ordered monolayers of a tetralactam macrocycle (TLM), i.e. the rotaxane wheel, are prepared on a Au(111) surface. Then, semiaxles (SA) are deposited on top of these ordered TLM layers at ca. 140 K. In solution, the SA molecule threads into the TLM cavity by formation of three hydrogen bonds between the amide groups of both molecules. On the Au(111) surface, the scenario is similar, although different in detail due to geometric restrictions given by the underlying Au(111) surface and conformational energy barriers due to the confinement of the TLM geometry in the ordered monolayer structure. Three distinct and defined adsorption sites of the SA molecules with respect to the TLM molecules exist. Notably, the population of these sites is assisted by interaction with the STM tip. Two sites are compatible with a structural model, in which the tail of the SA molecule binds into the TLM cavity, in one case with three H bonds, one to the terminal NH2 group of the SA and two to the central amide group. This SA—TLM adsorption complex formed at low temperatures is metastable and dissociates at higher temperatures. These results demonstrate the possibility to study intermolecular complex formation by STM.