The neuromuscular junction (NMJ) in Drosophila larvae serves as an important model system to study synaptic vesicle recycling, plasticity, development, and organelle trafficking in nerve terminals. Here we provide a protocol that reliably localizes proteins subcellularly in this system in combination with good preservation of the ultrastructure of the NMJ. In this protocol, the NMJs are cut open to increase access of reagents to the interior of the synapse, stained with primary antibodies followed by secondary antibodies conjugated to Fluoronanogold particles to allow selection of the best-stained areas for electron microscopic (EM) analysis using confocal microscopy. To improve visualization at the EM level, the nanogold particles are silver enhanced. Good penetration of the antibodies in this protocol allows for a three-dimensional reconstruction of the labeling pattern from serial ultrathin sections. To explore the utility and resolution of this approach, we determined the ultrasturcutral localization of the cell adhesion molecule Fascicline II (Fas II) at rest using an antibody raised against the cytoplasmic epitope. We observed that Fas II-ir is accumulated at the cytoplasmic surfaces of pre- and postsynaptic elements of the synapse at equal distances from the membranes, thus supporting the model that it has a similar orientation in these opposing synaptic structures. Fas II-ir delineates active zones and is compartmentalized from CSP, Dap160, and Bruchpilot immunolabeling. We conclude that Fas II immunolabeling in Drosophila can be reliably used to evaluate changes in the size of the active zone region at rest in genetic and functional experiments.