This is the first report of bioreactive self-assembled monolayers, covalently bound to atomically flat silicon surfaces and capable of binding biomolecules for investigation by scanning probe microscopy and other surface-related assays and sensing devices. These monolayers are stable under a wide range of conditions and allow tailor-made functionalization for many purposes. We describe the substrate preparation and present an STM and SFM characterization, partly performed with multi-walled carbon nanotubes as tapping-mode super-tips. Furthermore, we present two strategies of introducing in situ reactive headgroup functionalities, One method entails a free radical chlorosulfonation process with subsequent sulfonamide formation. A second method employs singlet carbene-mediated hydrogen-carbon insertion of a heterobifunctional, amino-reactive trifluoromethyldiazirinyl crosslinker, We believe that this new substrate is advantageous to others, because it (i) is atomically flat over large areas and can be prepared in a few hours with standard equipment, (ii) is stable under most conditions, (iii) can be modified to adjust a certain degree of reactivity and hydrophobicity, which allows physical adsorption or covalent crosslinking of the biological specimen, (iv) builds the bridge between semiconductor microfabrication and organic/biological molecular systems, and (v) is accessible to nanopatterning and applications requiring conductive substrates. (C) 1997 Academic Press.