Electrosurface phenomena at thin polymer films utilized in the preparation of biosensors have been studied by use of the Microslit Electrokinetic set-up. For the investigated polymer layers (aminodextran, carboxylated dextran, diaminopoly(ethylene glycol), dicarboxypoly(ethylene glycol), biotinylated poly(ethylene glycol), and streptavidin on biotinylated poly(ethylene glycol), the charge formation in aqueous electrolyte solutions was found to depend on the pH value, that is, OH- and H3O+ are the charge determining ions. The isoelectric points obtained from zeta potential versus pH plots could be utilized to draw conclusions on the introduction of acidic or basic groups and on the degree of molecular surface coverage, respectively. The hydrodynamically mobile charge reflected by the magnitude of the zeta potential contributed to only about 6% or less of the total surface conductivity of the polymer layers. The experimental determination of the total surface conductivity was found to provide valuable information on structural features of biosensor interfaces in aqueous environments.