DNA binding proteins of the winged helix family contain a conserved 110 amino acid region, the fork head/HNF-3 domain. Three members of the recently described XFD (Xenopus fork head domain related) multigene family in the frog Xenopus laevis that contain this DNA-binding domain have been studied. We determined the in vitro DNA recognition sequences by means of two independent methods: PCR supported site selection with degenerated deoxyoligonucleotides and affinity chromatography of genomic Xenopus DNA fragments. In contrast to a remarkable sequence divergence within their protein sequence of the fork head domains, all three proteins share a similar 7 bp DNA target motif. The protein-DNA interaction has been studied by means of DMS interference and hydroxyl radical footprinting. A region of 18 bp encompassing the 7 bp target motif is sufficient to confer binding and specificity. The specificity of binding could be attributed on the DNA level to residues located 5' to the 7 bp core region, and on the protein level most likely to a region within the first half of the fork head domain. The possible role of specific nucleotides within the target site in binding the protein is discussed in the context of the current crystal structure of the complex of this domain with DNA.