Visualization of specific transsynaptic neural pathways is an indispensable technique for understanding the relationship between structure and function in the nervous system. Here, we demonstrate the application of the wheat germ agglutinin (WGA) transgene technique for tracing transsynaptic neural pathways in Drosophila. The intracellular localization of WGA was examined by immunoelectron microscopy. WGA signals were detected in granule-like structures in both the outer photoreceptor cells expressing WGA and the second-order laminar neurons. Misexpression of tetanus toxin (TNT), which inactivates N-synaptobrevin, in the outer photoreceptor cells resulted in the elimination of on/off transients in electroretinogram (ERG) recordings and in a great reduction in WGA transfer into laminar neurons, suggesting that anterograde WGA transsynaptic transfer is dependent mainly on synaptic transmission. Retrograde WGA transfer was also detected upon its forced expression in muscle cells. WGA primarily expressed in muscle cells was taken up by motoneuron axons and transported to their cell bodies in the ventral nerve cord, suggesting that WGA can trace motoneuronal pathways in combination with the muscle-specific GAL4 driver. Thus, the GAL4/UAS-WGA system should facilitate the dissection of the Drosophila neural circuit formation and/or synaptic activity in various regions and at various developmental stages.