We present the first structural evidence supporting the hypothesis that the binding specificity of the winged helix DNA binding motif is mediated by residues adjacent to the alpha-helix (H3), the moiety which is primarily involved in the interaction with DNA. Using NMR to determine secondary structural elements of a winged helix family member, Genesis (formerly HFH-2), and comparing these with those found in the X-ray crystal structure of the HNF-3gamma/DNA complex [Clark, K. L., Halay, E. D., Lai, E., & Burley, S. K. (1993) Nature 364, 412-420], we show that the major differences observed occur for H3 and the region immediately prior to this. H3 in Genesis is slightly shorter than in HNF-3gamma and, in addition, we observe an extra small helix (H4) in the region between H2 and H3 which is not found in the HNF-3gamma/DNA complex. This is significant as it has been shown previously [Overdier, D. G., Porcella, A., & Costa R. H. (1994) Mol. Cell. Biol. 14, 2755-2766] that the DNA-binding specificity is influenced by amino acid residues in this region.