The purines ATP, ADP, and adenosine are important extracellular signaling agents. Analysis of purinergic signaling has been slowed by lack of direct methods for measurement of purine release in real-time during physiological activity. We have previously reported microelectrode biosensors for adenosine, but similar sensors for ATP have remained elusive. We now describe an ATP biosensor formed by coating a Pt microelectrode with an ultrathin biolayer containing glycerol kinase and glycerol-3-phosphate oxidase. It responds rapidly (10-90% rise time <10 s) and exhibits a linear response to ATP over the physiologically relevant concentrations of 200 nM-50 microM and is very sensitive approximately 250 mA.M(-1).cm(-2). By including phosphocreatine kinase in the biolayer, we can optionally amplify the ATP signal and also make the sensor sensitive to external ADP. We have used our sensors to make the first demonstration that ATP is released from spinal networks in vivo during locomotor activity.