We report the identification of a compound whose K+-induced Ca2+-dependent release in rat cerebellar slices was reduced following climbing fibre deprivation by 3-acetylpyridine (3-AP) treatment. Based on HPLC retention time, UV absorption spectrum, and mass spectrometry, this compound was identified as adenosine. The K+-induced, Ca2+-dependent release of adenosine was subsequently quantified in control and 3-AP-treated rats. It decreased by 60 - 70% in both the cerebellar vermis and hemispheres following climbing fibre deprivation, while 3-AP treatment had no effect on adenosine release in the cerebral cortex. Inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP and GMP decreased basal and stimulated efflux of adenosine in the cerebellum by 50 - 60%, indicating that a significant proportion of adenosine was derived from the extracellular metabolism of released nucleotides. Taken with the reports of other groups on adenosine in cerebellum, these results suggest that climbing fibre activity increases the extracellular level of adenosine, probably through the metabolism of released nucleotides. This adenosine could then cause presynaptic inhibition of the release of the parallel fibre transmitter, which is presumably glutamate. This may account for the climbing fibre-evoked depression of Purkinje cell sensitivity to parallel fibre input.