Developmental and tissue-specific transcription from the Adh distal promoter is regulated in part by the Adh adult enhancer, located 450 to 600 bp upstream from the distal RNA start site. We have characterized four proteins (DEP1 to DEP4), present in Drosophila tissue culture cell nuclear extracts, which bind to this enhancer. DEP1 and DEP2 bind to a positive cis-acting element (-492 to -481) and share nucleotide contacts. A small linker replacement deletion mutation, which disrupts the overlapping DEPl-and DEP2-binding sites, reduces Adh distal transcription in an alcohol dehydrogenase (ADH)-expressing cultured cell line, in the adult fat body (the major tissue of ADH expression), as well as in some but not all adult tissues where ADH is normally expressed. This enhancer element contains an imperfect palindromic sequence similar to steroid hormone receptor superfamily response elements. Binding-site screening of a A gtll expression library has identified the steroid receptor superfamily member fushi tarazu factor 1 (FTZ-F1) as a protein that binds to this site. Anti-FTZ-Fl antibodies have identified DEP1 as FTZ-F1. DEP2 also binds to the FTZ-F1 site from the fushi tarazu zebra element, suggesting that DEP2 may also be a steroid receptor superfamily member. Our results raise the possibility that Adh regulation in certain adult tissues involves a hormone-mediated pathway. Because DEP1 (FTZ-F1) and DEP2 contact some of the same nucleotides within the positive cis element, it is unlikely that they can bind simultaneously. Such alternative binding may play a role in the tissue-specific and developmental transcription of Adh.The alcohol dehydrogenase (ADH) gene (Adh) of Drosophila melanogaster is transcribed from two promoters, the proximal (larval) and distal (adult) (7). Developmental and tissue-specific transcription from the distal promoter has been well characterized (36, 54) and involves specific interactions between the promoter and enhancer (3,11,12). The compact arrangement of the distal promoter and enhancer (see Fig. la for the organization of the Adh gene) and the existence of cell lines that differ in their ability to utilize the adult enhancer (3) provide an excellent system to study specific factors which control developmental and tissuespecific transcription.Several approaches have been used to study distal promoter regulation, including germ line transformation, transient transfection, chromatin structure analysis, and biochemical characterization of DNA-binding proteins. P-element-mediated germ line transformation has identified cis elements required for correct distal promoter expression (11-13, 49) and defined the distal (adult) enhancer. Chromatin structure analysis has suggested that differential transcription from the distal promoter is the result of differences in DNA-protein interactions (5, 9) and may involve differences in the positioning of nucleosomes (18, 30a).Biochemical identification of proteins which bind to the promoter region has resulted in the purification of 26), a protein whic...