We have isolated and sequenced the pseudo-allelic versions of FoxD5 genes in Xenopus laevis, xlFoxD5a and xlFoxD5b, and the xtFoxD5 gene of Xenopus tropicalis. These genes show a highly conserved structure, they are composed of only one exon, and they exhibit a high degree of sequence conservation within their flanking sequences. The X. tropicalis gene is expressed like its X. laevis orthologues in progenitor cells of the neural floor plate. Serial deletions of the 5'-and 3'-flanking region in combination with reporter gene assays demonstrate that a distal and a proximal upstream element as well as a downstream-located enhancer do mainly contribute to transcriptional activity. The downstream enhancer cooperates with the proximal upstream element and also contributes to the spatial expression. Transgenic animals express enhanced green fluorescent protein in a spatial pattern like xlFoxD5b, when the 5'- and 3'-flanking regions of the xlFoxD5b gene are used to direct transgene expression. (C) 2004 Elsevier Inc. All rights reserved.