Force-distance studies suggest that electrostatic forces play a dominant role in the resistance of oligo(ethylene glycol) (OEG) terminated self-assembled monolayers (SAMs) to adsorption from aqueous solution, whereas for end grafted poly(ethylene glycol) (PEG) coated surfaces inertness is explained by a steric repulsion effect. To study the role of electrostatics in the repulsive forces measured on methoxytri(ethylene glycol) (EG(3)OMe) terminated alkanethiolate SAMs on gold surfaces, their electrokinetic potentials and charging properties were determined with the microslit electrokinetic setup (MES) using rectangular microchannels formed by SAM-covered sample carriers. The zeta potentials measured on the (EG(3)OMe) SAMs, both on gold and on glass substrate, indicate preferential hydroxide ion adsorption from the aqueous phase resulting in a net negative surface charge at pH > 4. Calculated charge densities agree well with the results from force-distance studies. We also studied the electrokinetic properties of poly(ethylene glycol) (PEG) SAMs (HS(CH2)(11)(OCH2CH2)(44)-OCH3). All these films show a negative zeta potential in neutral and alkaline solutions which decreases with increasing packing density and optical film thickness, reflecting the changes in frictional properties of the PEG chains with increasing coverage. An explanation why the negatively charged (EG(3)OMe) surfaces exhibit repulsive electrostatic forces in aqueous solution, but other negatively charged surfaces do not, is proposed.