Serotonin (5-HT) receptors receptors located close to the synaptic contacts modulate synaptic transmission through a variety of pre-and postsynaptic processes. To investigate the dimension of the functional 5-HT domains and to determine the underlying signaling pathways, cultured hippocampal neurons were patch clamp recorded. Synaptic inputs were standardized using ionophoretic glutamate application inducing inward currents (GluPSC) comparable to synaptic EPSCs. A pressure system allowed a controlled and circumscribed application of pharmacological substances and activation of local receptors. Local application of the 5-HT4 receptor agonist RS67333 (200mM) caused a reversible reduction of GluPSC amplitudes (65.9%) and an increase of the decay time constant (171.7%). Higher concentrations of the 5-HT4 agonist (500mM) induced a strong inward current or a depolarization of the membrane potential. Controversially, bath application of the selective 5-HT4 receptor antagonist GR113808 induced also a depolarization and in addition a termination of spontaneous synaptic activity. The latter findings provide evidence for a constitutive 5-HT4 activity. Taken together, we demonstrate that different activation levels of the 5-HT4 receptors have distinct concequences in synaptic transmission processes.