To identify cholinergic neurons, we isolated a choline acetyltransferase (Ci-ChAT) gene from Ciona intestinalis by PCR methods. In the cloning process, we also obtained the gene encoding the vesicular acetylcholine transporter (Ci-vAChTP). These two genes shared the same 5'-UTR sequence as well as similar expression patterns. In both cases, the gene expression was first detected by whole-mount in situ hybridization in the anterior-dorsal region of the caudal nerve cord at the early tailbud stage. In the larva, the expression was seen in several cells of the visceral ganglion. These results suggest that ascidian larval motor neurons exist in the visceral ganglion.