A glucose oxidase (GOx) electrode was used to determine glucose amperometrically in acetonitrile-water (90 + 10% v/v) using (carboxycyclopentadienyl)cyclopentadienyliron [ferrocenemonocarboxylic acid (FMCA)] as a soluble electron-transfer mediator. In this predominately organic phase, FMCA exhibits reversible Nernstian kinetics. The response of the glucose sensor (indicated by the catalytic current, IK) in acetonitrile (IK= 96 µA) was almost double its response in phosphate buffer (IK= 55 µA) or piperazine-N,N′- bis(2-ethanesulfonic acid)(PIPES) buffer (IK= 57 µA). The ratio of the anodic peak current in the presence, to that in the absence, of enzymic activity (IK/ID), obtained from analyses of cyclic voltammograms, was used as an index of catalytic efficiency of the biosensor. The values of this parameter were found to be 7.1, 7.0 and 6.9 in phosphate buffer, acetonitrile and PIPES buffer, respectively. Large steady-state currents of the sensor in acetonitrile are reminiscent of responses usually obtained by the perturbation of the carbohydrate moiety shielding the active redox centre of GOx induced by special immobilization strategies. This phenomenon was explained on the basis of the energetically favourable concomitant desorption of GOx-active site-bound water, by the hydrophilic acetonitrile, on glucose binding. This solventenhanced activity shows that organic-phase biosensors can be constructed by simple methods for any enzyme, provided that the critical amount of water needed for optimum response is determined.