1. Nitric oxide (NO) release following repetitive electrical stimulation was studied in the molecular layer of rat cerebellar slices using electrochemical NO probes. 2. In parasagittal slices of the vermis, most Purkinje cells showed climbing fibre responses in response to white matter stimulation without accompanying NO release. 3. In frontal slices, parallel fibre volley potentials and NO release were elicited concurrently by parallel fibre stimulation. 4. The NO release following parallel fibre stimulation was not affected by blockers of non-NMDA, NMDA and metabotropic glutamate receptors. 5. The NO release was reduced significantly (P < 0.001) to 29% of the control level after climbing fibre deafferentation with 3-acetylpyridine treatment. 6. The rate of NO release was roughly proportional to the second or third power of the stimulus frequency, and to the third power of the extracellular Ca2+ concentration. 7. The rate of NO release was not affected by nicardipine (10 microM). It was reduced to 87 +/- 4% (n = 5, mean +/- S.E.M.) of the control level by omega-conotoxin GVIA (0.3 microM), and to 18 +/- 4% (n = 4) by omega-agatoxin IVA (0.3 microM). 8. Tetanic parallel fibre stimulation potentiated NO release by 24 +/- 5% (n = 5). 9. These data indicate that NO is derived mainly from parallel fibres. The relationship between NO release and cerebellar synaptic plasticity is discussed.