Simultaneous microelectrode recording from two individual synaptically connected neurons enables the direct analysis of synaptic transmission and plasticity at a minimal synaptic connection. We have recorded from pairs of CA3 pyramidal neurons in organotypic hippocampal slices to examine the properties of long-term potentiation (LTP) at such minimal connections. LTP in minimal connections was found to be identical to the NMDA-dependent LTP expressed by CA3-CA1 synapses, demonstrating this system provides a good model for the study of the mechanisms of LTP expression. The LTP at minimal synaptic connections does not behave as a simple increase in transmitter release probability, because the amplitude of unitary EPSCs can increase several-fold, unlike what is observed when release probability is increased by raising extracellular calcium. Taking advantage of the relatively short axon connecting neighboring CA3 neurons, we found it feasible to introduce pharmacological agents to the interior of presynaptic terminals by injection into the presynaptic soma and have used this technique to investigate presynaptic effects on basal transmission and LTP. Presynaptic injection of nicotinamide reduced basal transmission, but LTP in these pairs was essentially normal. In contrast, presynaptic injection of H-7 significantly depressed LTP but not basal transmission, indicating a specific role of presynaptic protein kinases in LTP. These results demonstrate that pharmacological agents can be directly introduced into the presynaptic cell and that a purely presynaptic perturbation can alter this plasticity.