We use self- and directed assembly to pattern organic monolayers on the nanometre scale. The ability of the scanning tunnelling microscope to obtain both nanometre-scale structural and electronic information is used to characterize patterning techniques, to elucidate the intermolecular interactions that drive them and to probe the structures formed. We illustrate three successful approaches: (1) phase separation of self-assembled monolayers by terminal and internal functionalization, (2) phase separation of self-assembled monolayers induced by post-adsorption processing and (3) control of molecular placement by insertion into a self-assembled monolayer. These methods demonstrate the possibilities of patterning films by exploiting the intrinsic properties of the molecules. We employ these methods to prepare matrix-isolated samples to probe molecular electronic properties of single and bundled molecules.