Synapses play a fundamental role in the processing of information in a neuronal network by mediating communication between neurons. To monitor presynaptic and postsynaptic structures simultaneously in living neurons, we developed double transfection method on the basis of calcium phosphate coprecipitation and introduced two fluorescent reporters into cultured neurons. The optimized protocol allowed different populations of neurons to be visualized by different color fluorescences, and presynaptic and postsynaptic structures to be observed in the same field of view of a microscope.