The tyramine receptor (TyrR) is a G protein-coupled receptor for trace amines, cloned in Drosophila melanogaster, and claimed to be either an octopamine receptor or a tyramine receptor. We previously reported that in the larval neuromuscular junctions, the modulatory effect on the excitatory junction potentials of tyramine is distinctly different from that of octopamine. The effect of tyramine but not of octopamine was selectively abolished in the TyrR mutant hono, suggesting that this gene encodes a receptor for tyramine, and not for octopamine. We examined whether there was a gene-dosage effect of this tyramine modulation using combinations of hono, deficiency (Df) and wild-type alleles. The tyramine effect was observed in hono heterozygotes (+/hono), which showed intermediate levels of response, but was not seen in +/Df or hono/Df hemizygotes. While these further suggest that tyramine is the true ligand, it is possible that the gene-dosage effect is only evident above some threshold of gene expression levels. Immunohistochemical staining using an anti-tyramine antibody identified tyramine-containing neurons in the larval central nervous system, some of which were distinct from the octopamine-containing neurons. Taken together, these results strongly suggest that tyramine functions as a neuromodulator.