This paper describes microfluidic networks based on glass microchannels with integrated insulated gate electrodes that are used to modify the zeta-potential at specific locations on the wall surface of the microchannels. This modification is used to control direction and size of the electro-osmotic flow (EOF) in the microchannels. A new microfabrication process was developed that compared to earlier work in our institute [R.B.M. Schasfoort, S. Schlautmann, J. Hendrikse, A. van den Berg, Science 286 (1999) 942] leads to mechanically more robust structures and better flow visualization in the gate region. In the new process sequence, the microfluidic chips were fabricated using deposition on a Pyrex glass substrate of a thin-film metal electrode, which was covered with a thin insulating film of silicon dioxide, followed by chemical mechanical polishing prior to bonding to a second glass plate to form closed microchannels. Electrical breakdown of the metal–insulator structures was measured and occurred at 9.6 ± 0.3 MV/cm. Experiments using fluorescent beads to visualize the flow patterns in the microchannel showed that the EOF is linearly dependent on the applied gate voltage. The average EOF could be stopped completely for longitudinal fields of 150 V/cm by applying a gate field of 1.7 MV/cm.