Aim: The purine adenosine is a potent CNS neuromodulator which plays a role in many physiological and pathological processes. Although much is known about the receptor- mediated actions of adenosine, it is still often unclear how adenosine is released into the extracellular space. It has been suggested that adenosine could arise from either the extracellular breakdown of ATP or by cytoplasmic translocation via transporters. Our results suggest that adenosine release is activity- dependent and has many features of direct release by exocytosis. Methods: Electrophysiology combined with microelectrode biosensor recordings from transverse rat cerebellar slices (postnatal days 21-24). Results: Trains of electrical stimuli, applied in the molecular layer, produce action potential and Ca2+ dependent adenosine release. Sufficient adenosine is released to inhibit parallel fiber- Purkinje cell synaptic transmission. Adenosine release is modulated by activators of the presynaptic receptors found on parallel fibre terminals (GABAB, mGluR4 and A1) strongly supporting a requirement for parallel fibre activation. Since no ATP could be detected, and inhibitors of ecto-ATPases had no effect on adenosine release, we suggest that adenosine is directly released. Low concentrations of 4-AP produced an increase in adenosine release which correlated with the broadening of the parallel fibre action potential. Under such conditions, a single action potential was sufficient to release adenosine. By varying the frequency of action potentials we have investigated the properties of adenosine release. Conclusions: Our data suggests that adenosine release has many of the properties of conventional transmitter release and plays an important role in controlling neural activity. Funded by the MRC.