twist is one of the earliest expressed zygotically active genes required for dorsal-ventral pattern formation in the Drosophila embryo. Genetic studies suggest that this gene is activated in the ventral part of the blastoderm by maternally expressed dorsal gene product. Using P-element-mediated germ-line transformation, we have mapped a small (260 bp) dorsal-dependent ventral activator region (VAR) in the 5'-flanking region of the twist promoter that can direct the early ventral expression of a heterologous promoter. The VAR contains binding sites for a number of proteins present in extracts of Drosophila embryos. One of these sites bears homology to known binding sites for the dorsal transcription factor and is specifically bound by bacterially expressed dorsal protein. Furthermore, a 37-bp deletion that removes the dorsal-binding sequences abolishes the ventral-specific activity of the twist promoter constructs. Our data also show that additional sequences within the VAR are required to render the dorsal-binding sites functional. Finally, reverse genetic and biochemical data suggest that the transcription factor, encoded by the zeste gene may help control the overall level, if not the pattern, of twist expression.