Retinoic acid regulates the spatial pattern of gene expression mainly in the epidermis in the protochordate ascidian, Ciona intestinalis. Our previous study characterized the enhancer element responsible for the activation of the retinoic acid receptor (RAR) in the dorsal midline epidermis (DME). In the present study, detailed analysis identified two small sequence elements required for the transcriptional activation in the DME. Deletion of either one of these sequences resulted in suppression of the DME enhancer, suggesting that both sites are necessary. The nucleotide sequences of these two sites were similar to the consensus recognition sequences for the Msx and Sox transcription factors, respectively. These transcription factors are expressed in the DME lineage blastomeres (b7.9 and b7.10 blastomere pairs of bilaterally symmetrical embryos of this species) at the 64-cell stage. Electrophoretic mobility shift assays revealed that recombinant Msxb and SoxB1 proteins specifically bound to these sequences. These results suggest that Msxb and SoxB1 synergistically activate the enhancer in the DME.