Various techniques have been applied for the functional analysis of synaptic transmission in cultured neurons. Here, we describe a method of studying synaptic transmission in neurons cultured at high-density from different brain regions such as the cortex, striatum and spinal cord. We use postsynaptic whole-cell recordings to monitor synaptic currents triggered by presynaptic action potentials that are induced by brief stimulations with a nearby extracellular bipolar electrode. Pharmacologically isolated excitatory or inhibitory postsynaptic currents can be reliably induced, with amplitudes, synaptic charge transfers, and short-term plasticity properties that are reproducible from culture to culture. We show that the size and kinetics of pharmacologically isolated inhibitory postsynaptic currents triggered by single action potentials or stimulus trains depend on the Ca²⁺ concentration, temperature and stimulation frequency. This method can be applied to study synaptic transmission in wildtype neurons infected with lentiviruses encoding various components of presynaptic release machinery, or in neurons from genetically modified mice, for example neurons carrying floxed genes in which gene expression can be acutely ablated by expression of Cre recombinase. The preparation described in this paper should be useful for analysis of synaptic transmission in inter-neuronal synapses formed by different types of neurons.