Extracellular application of protein kinase inhibitors was used to examine the role of calcium/calmodulin-dependent protein kinase II (CaM-KII) in synaptic transmission in the CA1 region of rat hippocampus. Bath application of the broad spectrum, membrane permeable kinase inhibitor H7 (250 microM) decreased excitatory synaptic responses elicited in hippocampal slices. Whereas H7 inhibits several protein kinases and has non-specific effects, several synthetic peptides have been developed as specific inhibitors of CaM-KII. Using in situ phosphorylation in hippocampal slices, we demonstrate that extracellular application of synthetic peptide inhibitors of CaM-KII preferentially suppresses the phosphorylation of synapsin I at the CaM-KII specific site. This suppression was not reversed by the application of a calcium ionophore indicating the decrease in phosphorylation does not result only from blockade of presynaptic calcium influx. Thus, it appears the peptides gain access to intracellular compartments and retain their inhibitory properties. Further, we found that extracellular application of these peptide inhibitors decreased excitatory synaptic responses elicited in the CA1 region of hippocampal slices with relative potencies consistent with their ability to block CaM-KII activity in vitro. Peptide application did not alter the input resistance of postsynaptic cells nor responses elicited by glutamate iontophoresis. These results suggest that CaM-KII activity, possibly through phosphorylation of presynaptic synapsin I, is required for sustained synaptic transmission at mammalian synapses.