Surface reactions were employed to introduce a variety of functional groups in self-assembled monolayers (SAMs) on hydroxylated surfaces such as glass and oxidized silicon wafers. The resulting layers were fully characterized by wettability studies, ellipsometry, Brewster angle infrared and x-ray photoelectron spectroscopy, UV-visible absorption and fluorescence measurements. Based on these measurements, it was concluded that the steric hindrance of the monolayer hampered the surface reactions, which generally led to sub-monolayer coverages. Detailed information concerning the interactions amongst adsorbates was obtained from fluorescence measurements. The characteristic excimer emission of pyrene-functionalized layers showed a large dependence on the solvent that was in contact with the SAM. Furthermore, efficient energy transfer could be observed in mixed monolayers that contained both fluorescein and lissamine groups.