The innate immunity of ascidian hemocytes is considered to be a prototype of that in vertebrates. In this study, we identified as many transcripts as possible that were expressed specifically in hemocytes of Ciona intestinalis, a ubiquitous species of ascidian. Using a large-scale whole-mount in situ hybridization (WISH) technique and young adult specimens of C. intestinalis, 34 such transcripts were identified. Three of these appeared to encode immunity-related polypeptides, whereas 23 encoded hypothetical and/or new genes. Interestingly, different sets of transcripts appeared to be expressed in different subsets of hemocytes, as revealed by double-colored WISH. The 34 genes were categorized into two major subgroups based on their expression patterns during the C. intestinalis life cycle. Based on the gene expression profiles, we speculate that C. intestinalis hemocytes may exert more pleiotropic effects in immunity than previously believed.