Cerebellar long-term potentiation (LTP) is a persistent increase in the strength of the granule cell-Purkinje neuron synapse that occurs after brief stimulation of granule cell axons at 2-8 Hz. Previous work has indicated that cerebellar LTP induction requires presynaptic Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA. The evidence implicating PKA has come from bath application of drugs during LTP induction, an approach that does not distinguish between PKA activation in the presynaptic or postsynaptic cell. Although bath application of PKA inhibitor drugs (KT5720, Rp-8CPT-cAMP-S) blocked LTP induction in granule cell-Purkinje neuron pairs in culture, selective application to granule cell or Purkinje neuron somata via patch pipettes did not. We hypothesized that presynaptic PKA activation is required for LTP induction but that drugs applied to the granule cell soma cannot diffuse to the terminal within this timescale. To test this hypothesis, we transfected cerebellar cultures with an expression vector encoding a peptide inhibitor of PKA [Rous sarcoma virus (RSV)-protein kinase A inhibitor (PKI)]. Transfection of RSV-PKI into presynaptic granule cells, but not postsynaptic Purkinje neurons or glial cells, blocked LTP induction produced by either synaptic stimulation or an exogenous cAMP analog. An expression vector encoding a control peptide with no PKA inhibitory activity was ineffective. These results show that induction of cerebellar LTP requires a presynaptic signaling cascade, including Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA, and argue against a requirement for postsynaptic Ca signals or their sequelae.