The ascidian larva is an excellent model for studies of the functional organization and neuronal circuits of chordates due to its remarkably simple central nervous system (CNS), comprised of about 100 neurons. To date, however, the identities of the various neurons in the ascidian larva, particularly their neurotransmitter phenotypes, are not well established. Acetylcholine, GABA, and glycine are critical neurotransmitters for locomotion in many animals. We visualized putative cholinergic neurons and GABAergic/glycinergic neurons in the ascidian larva by immunofluorescent staining using antibodies against vesicular acetylcholine transporter (VACHT) and vesicular GABA/glycine transporter (VGAT), respectively. Neurons expressing a cholinergic phenotype were found in the brain vesicle and the visceral ganglion. Five pairs of VACHT-positive neurons were located in the visceral ganglion. These putative cholinergic neurons extended their axons posteriorly and formed nerve terminals proximal to the most anterior muscle cells in the tail. VGAT-positive neurons were located in the brain vesicle, the visceral ganglion, and the anterior nerve cord. Two distinct pairs of VGAT-positive neurons, bilaterally aligned along the anterior nerve cord, extended axons anteriorly, near to the axons of the contralateral VACHT-positive neurons. Cell bodies of the VGAT-positive neurons lay on these nerve tracts. The neuronal complex, comprising motor neurons with a cholinergic phenotype and some of the GABA/glycinergic interneurons, has structural features that are compatible with a central pattern generator (CPG) producing a rhythmic movement of the tail. The simple CPG of the ascidian larva may represent the ancestral state of the vertebrate motor system.