AbstractGlutamate is a major neurotransmitter in the excitatory synapses of both vertebrate and invertebrate nervous systems and is involved in many neural processes including photo‐, mechano‐, and chemosensations, neural development, motor control, learning, and memory. We identified and characterized the gene (Ci‐VGLUT) encoding a member of the vesicular glutamate transporter subfamily, a specific marker of glutamatergic neurons, in the ascidian Ciona intestinalis. The Ci‐VGLUT gene is expressed in the adhesive organ, the epidermal neurons, and the brain vesicle, but not in the visceral ganglion. The Ci‐VGLUT promoter and an anti‐Ci‐VGLUT antibody were used to analyze the distribution and axonal connections of prospective glutamatergic neurons in the C. intestinalis larva. The green fluorescent protein (GFP) reporter driven by the 4.6‐kb upstream region of Ci‐VGLUT recapitulated the endogenous gene expression patterns and visualized both the cell bodies and neurites of glutamatergic neurons. Papillar neurons of the adhesive organs, almost all epidermal neurons, the otolith cell, and ocellus photoreceptor cells were shown to be glutamatergic. Each papillar neuron connects with a rostral epidermal neuron. Axons from rostral epidermal neurons, ocellus photoreceptor cells, and neurons underlying the otolith terminate in the posterior brain vesicle. Some caudal epidermal neurons also send long axons toward the brain vesicle. The posterior brain vesicle contains a group of Ci‐VGLUT‐positive neurons that send axons posteriorly to the visceral ganglion. Our results suggest that glutamatergic neurotransmission plays a major role in sensory systems and in the integration of the sensory inputs of the ascidian larva. J. Comp. Neurol. 508:249–263, 2008. © 2008 Wiley‐Liss, Inc.