Patterned self-assembled monolayers (SAMs) were formed using the scanning electrochemical microscope (SECM). The procedures is based on the local electrochemical desorption of an alkanethiolate monolayer by applying a 5 kHz square-wave voltage of 2 V (peak-to-peak) to a two-electrode configuration consisting of an ultramicroelectrode (UME) of 10 mum diameter placed about 5 mum above a macroscopic SAM-covered gold electrode. Desorption occurs on well-defined regions with a diameter of (12.8 +/- 2.8) mum. These regions of bare gold are able to chemisorb a omega -functionalized thiol or disulfide such as cystamine to form patterns of amino-terminated surfaces. Functional proteins can be coupled to the amino groups present at the modified regions of the monolayer. This approach was demonstrated by imaging the activity of horseradish peroxidase bound to the patterned SAMs in the generation-collection mode of the SECM. A considerable improvement of the procedure could be achieved by performing the desorption in a solution containing a millimolar concentration of the co-functionalized thiol/disulfide ensuring effective refilling of the monolayer by the desired molecules and hence high concentration of the immobilized proteins. The method is discussed with respect to prospective application in the field of chip-based bioanalytical assays.