Miniaturized electrochemical biosensors for glucose were constructed, using recessed gold microelectrodes (diameter 350 to 425 mu m) and ultramicroelectrodes (diameter less than 25 mu m) as the base sensors. The gold was a substrate for chemically attached thiolated glucose oxidase containing adsorbed hydroquinonesulfonate, which served as a mediator of electron transfer between the electrode surface and the enzyme. The chemically modified electrodes were coated with Nafion(R) to reduce the effects of anionic interferences, such as ascorbic acid, and were tested in sodium phosphate buffered glucose solutions (pH 7.4) at 25 degrees C by cyclic voltammetry and steady-state amperometry at positive potentials suitable for glucose detection. Cyclic voltammetry confirmed that the glucose oxidase and hydroquinonesulfonate were both securely immobilized at the surface of the electrodes. The amperometric t(90) response times for the ultramicroelectrodes and microelectrodes were under 20 and 100s respectively. The current response of both electrode types was linear up to glucose concentrations of 50 mM, and the response to ascorbic acid at physiological concentrations was reduced by Nafion(R) by 53.3%. Key advantages of this electrode are that it is miniaturized for optimum in vivo analysis, it is unresponsive to variable oxygen tension, and its recess allows for increased response stability and faster response times.