Long-term potentiation (LTP) in the hippocampus is a possible mechanism for mammalian learning and memory in which protein kinases play critical roles. We have investigated the involvement of cyclic AMP-dependent protein kinase (PKA) in LTP by directly studying its activation. We developed an in vitro assay which is useful for selective and accurate measurement of stimulus-induced changes in PKA activity in hippocampal slices. PKA was transiently activated 2 and 10 min after delivery of LTP-inducing stimuli in area CA1 of the hippocampus. This activation did not persist during early or late phases of LTP, suggesting that the role of PKA is in the induction of LTP, not in its expression. LTP was not associated with any change in the total activity of PKA, consistent with activation by cyclic AMP, as opposed to an increase in the amount or efficacy of the enzyme. The LTP-associated activation of PKA required stimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, and bath application of NMDA was sufficient to activate PKA. Together, these results indicate that at the initiation of LTP, NMDA receptor stimulation leads to transient activation of PKA, and support a role for PKA in the induction of LTP.