Adenosine is an important signalling molecule involved in a large number of physiological functions. In the brain these processes are as diverse as sleep, memory, locomotion and neuroprotection during episodes of ischemia and hypoxia. Although the actions of adenosine, through cell surface G-protein coupled receptors, are well characterised, in many cases the sources of adenosine and mechanisms of release have not been defined. Here we demonstrate the activity-dependent release of adenosine in the cerebellum using a combination of electrophysiology and biosensors. Short trains of electrical stimuli delivered to the molecular layer in-vitro, release adenosine via a process that is both TTX and Ca2+ sensitive. As ATP release cannot be detected, adenosine must either be released directly or rapidly produced by highly localised and efficient extracellular ATP breakdown. Since adenosine release can be modulated by receptors that act on parallel fibre-Purkinje cell synapses, we suggest that the parallel fibres release adenosine. This activity-dependent adenosine release exerts feedback inhibition of parallel fibre-Purkinje cell transmission. Spike mediated adenosine release from parallel fibres will thus powerfully regulate cerebellar circuit output.