The oxygen dependence of a first generation amperometric biosensor was investigated in vitro and in vivo by monitoring its glucose response as a function of solution pO(2). The biosensor was a glucose oxidase (GOx) modified poly(o-phenylenediamine) coated Pt cylinder electrode (Pt/PPD/GOx) that has been designed for neurochemical analysis in vivo. Two types of oxygen probes were used: a self-calibrating commercial macroelectrode in vitro; and a carbon paste microelectrode in vivo. Calibrations in vitro showed that oxygen interference in the operation of Pt/PPD/GOx electrodes was minimal for concentrations of glucose (approximately 0.5 mM) and oxygen (approximately 50 microM) found in brain ECF. This observation was confirmed by simultaneous monitoring in vivo of brain glucose and oxygen in the awake rat. However, at levels of glucose normally found in peripheral tissues (approximately 5 mM), the oxygen dependence was severe. We conclude that the oxygen sensitivity of Pt/PPD/GOx biosensors does not preclude their reliable use in media containing low glucose levels, such as brain ECF.