The homeobox gene Not is highly conserved in Xenopus, chicken and zebrafish with an apparent role in notochord formation, which inspired the name of this distinct subfamily. Interestingly, Not genes are also well conserved in animals without notochord such as sea urchins, Drosophila or even Hydra, but appear to be highly derived in mammals. A search for homeobox genes in the placozoan Trichoplax adhaerens, one of the simplest organisms available today, revealed only two homeobox genes: a Not homologue and the previously described gene Trox-2, which is most similar to the Gsx subfamily of the Hox/ParaHox cluster genes. Not has a unique expression profile in Trichoplax. It is highly expressed in folds of intact animals and in the wounds of regenerating animals. The dynamic expression pattern of Trichoplax Not is discussed in comparison with the invariable expression pattern of Trox-2 and the putative secreted protein Secp1. The high sequence conservation of Not from Trichoplax to lower vertebrates, but not to mammals, represents a rare example of an apparent gene decay in the lineage leading to humans.