Xnot-2 is a homeobox gene expressed in Spemann's organizer. Here we present evidence that microinjection of synthetic Xnot-2 mRNA leads to the formation of notochord. Microinjection into the dorsal side of the Xenopus embryo results in greatly expanded notochords. Nearby somitic and prechordal mesoderm becomes recruited into these enlarged notochords, which also affect CNS patterning. Two early genes expressed in the developing notochord, chd and XFKH-1, are activated by Xnot-2. We conclude that gain-of-function of Xnot-2 promotes notochord formation.