ABSTRACT In Drosophila, two TGF-β growth factors, dpp and screw, function synergistically to subdivide the dorsal ectoderm into two embryonic tissues, the amnioserosa and dorsal epidermis. Previous studies have shown that peak dpp activity is required for the localized expression of zerknüllt (zen), which encodes a homeodomain transcription factor. We present evidence that zen directly activates the amnioserosa-specific expression of a downstream target gene, Race (Related to angiotensin converting enzyme). A 533 bp enhancer from the Race promoter region is shown to mediate selective expression in the amnioserosa, as well as the anterior and posterior midgut rudiments. This enhancer contains three zen protein binding sites, and mutations in these sites virtually abolish the expression of an otherwise normal Race-lacZ fusion gene in the amnioserosa, but not in the gut. Genetic epistasis experiments suggest that zen is not the sole activator of Race, although a hyperactivated form of zen (a zen-VP16 fusion protein) can partially complement reduced levels of dpp activity. These results suggest that dpp regulates multiple transcription factors, which function synergistically to specify the amnioserosa.