Micropatterns of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) have been formed on polycrystalline gold and glass surfaces using different soft-lithographic approaches. The patterns have been analyzed by noncontact scanning force microscopy. The activities of the immobilized patterns were probed with local resolution by scanning electrochemical microscopy. The following approaches have been tested: (i) microcontact printing of octadecanethiol on gold, followed by chemisorption of cystaminium chloride and incubation of a mixture of HRP and glutaraldehyde to form a patterned cross-linked and grafted enzyme gel; (ii) microcontact printing of octadecanethiol on gold followed by chemisorption of HRP into which an thiol group had been introduced by modification with S-acetylthioglycolic acid-N-succinimidyl ester; (iii) application of a mixture of GOx with N-(3-dimethylaminopropyl)-N‘-ethylcarbodiimide hydrochloride (EDAC) to a stamp and contact with an aminated glass surface; (iv) delivery of a mixture of GOx with EDAC through microscopic open channels in a block of poly(dimethylsiloxane) pressed against an aminated glass surface. Satisfying contrast in enzymatic activity and high local enzyme activities were achieved by the modification of the gold surfaces. A general advantage of this monolayer system is the speed with which the binding of thiols to gold occurs. Therefore, the patterning step can be carried out with short contact times and the enzyme incubation can be made under controlled conditions in a moisture chamber. Approach ii offers the special advantage of reducing the number of process steps to two (stamping and application of thiolated enzyme), making it suitable for building up more complex structures formed by a sequence of structuring steps.