Biosensors for glucose and lactate were produced by the immobilization of agarose membranes containing glucose oxidase and lactate oxidase respectively inside 2 pm high chambertype oxygen sensors. The well-defined microvolume elements of these chambers provided a reproducible method of generating a thin enzyme layer over the electrode area. The chambers also acted to anchor the enzyme membrane in place without the need for any surface modification. The extent of the enzyme reaction was monitored by the decrease in oxygen reduction current at the gold cathode, and the normalized current decrease related to the concentration of the substrate (glucose or lactate). Enzyme entrapment inside lowtemperature gelling agarose resulted in sensors with response times of approximately 10 s, but with calibration curves that were under enzyme kinetic control. Loss of response sensitivity with time was observed and was attributed primarily to enzyme diffusion out of the membrane. By casting a cellulose acetate membrane on top of the agarose-enzyme gel it was possible to improve both the stability of the sensors and their linear response range. This approach produced a lactate sensor with an upper limit of detection enhanced ten-fold to a limit of about 10 mM lactate, compared to a limit of about 1 mM in the absence of the cellulose acetate.