Phorbol esters, such as tetradecanoylphorbol 13-acetate (TPA), have been used extensively in studies of cerebellar long-term depression (LTD), based on the hypothesis that activated protein kinase C (PKC) directly mediates alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation. Here, we show that TPA-induced depression of synaptic transmission between granule cells and Purkinje cells in culture is mediated through activation of the MEK1/2-ERK1/2 pathway. Phosphorylation of ERK1/2 induced by TPA and co-application of high potassium and glutamate was greatly attenuated by preincubating Purkinje cells with the MEK1/2 (MAPK ERK kinase 1/2) inhibitor PD98059. TPA-induced depression of synaptic transmission between granule cells and Purkinje cells was attenuated by PD98059. The MEK1/2 inhibitor also suppressed declustering of the ionotropic glutamate receptor subunit 2/3 (GluR2/3) induced by TPA and co-application of high potassium and glutamate, even though phosphorylation of Ser880 of GluR2/3 was not inhibited significantly in the presence of PD98059. These results suggest that ERK1/2 plays an essential role in TPA-induced depression via regulation of GluR2/3 declustering at the synapse.