Gill slits, a series of openings in the pharyngeal epithelium, are characteristic features of the hemichordate and chordate body plans. In ascidians, these openings, called stigmata, are formed in the branchial sac during juvenile development. Multiple whole-mount in situ hybridization analyses based on approximately 1,500 genes expressed in Ciona intestinalis juveniles, identified 28 genes expressed predominantly in the stigmatal cells. Expression patterns of these stigmatal genes were classified into four different categories. On the basis of these findings, we have been able to show that the peripheral region of a stigma consists of at least three different regions. The expression of a Dlk1-like gene was detected in nonciliated cells during the stigma perforation and division and was maintained in the basal region of the elliptical stigma. Expression of meichroacidin, tektin A1, and tektin B1 orthologs during the differentiation of the ciliated stigmatal cells suggests that some of the molecular mechanisms involved in sperm differentiation might be recruited for the stigma development, or vice versa. Components of the cilia such as alpha-tubulin and rootletin were also expressed in the stigmatal cells. These genes might facilitate further analyses regarding the evolution of the branchial fissures and the development of the ascidian stigmata.