Cerebellar long-term depression (LTD), a candidate cellular mechanism of motor learning, is induced by conjunctive activation of parallel fibres and a climbing fibre. Previous studies have shown that combinatorial application of high potassium and glutamate (K/glu) to cultured cerebellar neurons can mimic this conjunctive stimulation of presynaptic fibres and induces the LTD of miniature excitatory postsynaptic current (mEPSC) amplitudes lasting for more than 24 h. The late phase of this LTD (LLTD, > 3 h) depends on de novo transcription induced by prolonged conditioning. Here, the role of Calcineurin in the LLTD induction was examined. Application of a Calcineurin inhibitor FK506 mimicked the effect of K/glu-treatment by decreasing mEPSC amplitudes for more than 24 h. FK506-induced depression, as well as the K/glu-induced LLTD, was blocked by inhibitors of either mRNA synthesis or Ca/Calmodulin dependent kinase. In addition, the FK506-induced depression and K/glu-induced LLTD occluded each other, suggesting that they share the same mechanism. On the other hand, misexpression of the constitutively active form of Calcineurin in the Purkinje neuron nucleus blocked the LLTD induction by the K/glu-treatment. These results suggest that Calcineurin is involved in the induction of LLTD as a negative regulator. Furthermore, it was found that trapping superoxide, which is increased by neuronal activity and inactivates Calcineurin, suppressed the LLTD induction. Taken together, these results suggest that the LLTD might be induced by down-regulation of Calcineurin activity through superoxide in cultured Purkinje neurons.