The even-skipped (eve) gene of Drosophila melanogaster is a crucial member of the pair-rule class of segmentation genes. We report here the characterization of a 16-kb region sufficient for all known aspects of eve expression and the rescue of an eve null mutation. We began by examining 45 kb surrounding the eve coding sequence for DNaseI hypersensitive sites and other transcription units. We find that the previously identified eve regulatory elements, those for early stripes 2, 3, and 7 and the late element, do not generate prominent hypersensitive sites. However, strong, constitutive DNaseI hypersensitive sites flank a 16-kb region, within which one developmentally regulated site is found at the eve promoter region. P-element transformation of this 16-kb domain into eve mutants rescues them to adult viability. This 16-kb domain contains regulatory elements for all known features of eve expression: the seven major blastoderm stripes, minor stripe expression during germ band extension, and later expression in the lateral mesodermal muscle precursor cells, in the central nervous system, adjacent to the invaginating proctodeum, and in a ring around the anal pad. We have begun a preliminary dissection of the 16-kb domain into its constituent regulatory elements. Other major findings include the following: (1) There is a second element for late stripe expression adjacent to the traditional late element. (2) A stripe element 3' of the gene interacts with the late element to give rise to the minor stripes seen in the even-numbered parasegments. (3) Expression in the proctodeum and anal pad is driven by sequences both 5' and 3' of the gene. (4) Expression in different sites in the central nervous system is driven by separable elements widely dispersed throughout 8 kb 3' of the gene.