AFX is a human forkhead transcription factor. Based on results from studies of the orthologous transcription factor DAF-16 in Caenorhabditis elegans, it was suggested that some of the metabolic defects in both type I and type II diabetes may be due to unregulated activity of AFX. In the present study, we report the high-resolution NMR solution structure of the DNA binding domain of AFX. It is the first structure of the DNA binding domain from a small subfamily of forkhead transcription factors (i.e., AFX, FKHR, FKHRL1, FKHRL1P1, and FKHRP1). Despite rather low sequence identity for a protein within the forkhead family, the structure is remarkably similar to those of the DNA binding domains of HNF3-gamma and FREAC-11, and to a lesser extent the DNA binding domain of Genesis which displays a slightly altered orientation of the DNA recognition helix. The high degree of structural similarity between the DNA binding domains of different forkhead transcription factors implies that the repositioning of helix 3, observed for Genesis, cannot be a general feature for modulation of the DNA binding specificity. Other mechanisms that could influence the DNA binding specificity are discussed.