To determine the functions of genes in distinct tissues during the development of Drosophila, it is often desirable to have genetic tools for targeted gene expression in restricted subsets of cells. Here, we report the identification of the enhancer trap line OK371-Gal4, which is expressed in a defined subset of neurons from embryonic stage 15 to adulthood. In the ventral nerve chord, it is expressed almost exclusively in motoneurons and in the brain in a limited number of neuronal clusters. The OK371 enhancer trap element is inserted in the proximity of the annotated gene CG9887, which encodes a Drosophila vesicular glutamate transporter (DVGLUT). In situ hybridization experiments using antisense probes against the mRNAs of DVGLUT and neighboring genes confirm that OK371-Gal4 detects an enhancer of DVGLUT. DVGLUT-specific antibodies detect its expression in identifiable motoneurons, which are known to be glutamatergic in Drosophila. DVGLUT initially appears in small cytoplasmic punctae in the somata of these motoneurons. As development proceeds, DVGLUT-positive particles are transported along motor axons and become concentrated at neuromuscular junctions (NMJs), where they colocalize with the synaptic vesicle marker synaptotagmin. We find that the DVGLUT-specific antibodies are valuable tools for the identification of motoneurons and other glutamatergic neurons. In addition, the OK371-Gal4 line can be used for the targeted expression of any gene in these cells. Given that vesicular glutamate transporters are essential for the uptake of the neurotransmitter glutamate into synaptic vesicles these tools provide a means to test gene function in these functionally important neurons.