The crystal structure of a sex-specific enhancer element is described at a resolution of 1.6 A. This 16-bp site, designated Dsx(A), functions in the regulation of a genetic switch between male and female patterns of gene expression in Drosophila melanogaster. Related sites are broadly conserved in metazoans, including in the human genome. This enhancer element is unusually rich in general regulatory sequences related to DNA recognition by multiple classes of eukaryotic transcription factors, including the DM motifs, homeodomain, and high mobility group box. Whereas free DNA is often crystallized as an A-form double helix, Dsx(A) was crystallized as B-DNA and thus provides a model for the prebound conformation of diverse regulatory DNA complexes. Sequence-dependent conformational properties that extend features of shorter B-DNA fragments with respect to double helical parameters, groove widths, hydration, and binding of divalent metal ions are observed. The structure also exhibits a sequence-dependent pattern of isotropic thermal B-factors, suggesting possible variation in the local flexibility of the DNA backbone. Such fluctuations are in accord with structural variability observed in prior B-DNA structures. We speculate that sites of intrinsic flexibility within a DNA control element provide hinges for its protein-directed reorganization in a transcriptional preinitiation complex.