Many chordate- and vertebrate-specific characteristics develop depending on retinoic acid (RA). Because the gene encoding the RA receptor exists only in chordates, RA function seems to be involved in chordate evolution. A cDNA microarray analysis of 9287 non-redundant cDNA clones was used to screen for RA target genes in the ascidian Ciona intestinalis. In addition, the spatial expression pattern of 94 candidate RA target genes was examined by in situ hybridization in RA-treated and control embryos. Strong RA-induced upregulation of Hox-1 and Cyp26 was observed, as is the case in vertebrates. In addition, a number of novel candidate target genes was identified. These included transcription factors and signaling molecules, suggesting that various differentiation and/or morphogenetic pathways are modulated by RA. The expression of cell adhesion molecules, cytoskeletal proteins and extracellular matrix components was affected by RA. Changes in the expression pattern of these genes may be a direct cause of abnormal morphogenesis of the anterior neural tissues. RA also affected the expression of genes that seemed to be involved in neuronal functions. Although obvious homeotic transformation has not been observed, the function of various neural cell types seemed to be impaired by RA. The microarray data are reliable and will contribute to comprehensive understanding of RA action in the development and evolution of chordates.