An amperometric enzyme sensor capable of operating at high temperatures was developed by utilizing a "wired" thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus. The response of the system was monitored through detection of the product of the enzymatic reaction, NADH, which was electrocatalytically reoxidized to NAD by a thermostable redox mediator, osmium (1,10-phenanthroline-5,6-dione)2-poly(4-vinylpyridine), at Eapp = +150 mVvs Ag/AgCl/KClsat. The enzyme was "wired" onto the surface of graphite electrodes by using an epoxy-based poly(ethylene glycol) diglycidyl ether cross-linker. The stability of the sensor at higher temperatures clearly surpassed the conventional system utilizing a mesophilic G6PDH (mG6PDH) from Leuconostoc mesenteroides. The mG6PDH-based system lost 26% of its response after 20 min at 50 degrees C. The response of the tG6PDH-based system remained unchanged under the same conditions. The tG6PDH-based system demonstrated excellent stability up to a temperature of 83 degrees C.