Fractionation of a nuclear extract derived from Drosophila tissue culture cells reveals the presence of multiple components involved in accurate transcription of both distal and proximal promoters of the alcohol dehydrogenase (Adh) gene. Transcription of deletion mutants indicates that a region between -24 and -85 upstream of the distal start site contains sequences required for RNA synthesis in vitro. Moreover, sequences that overlap this same upstream control region are specifically bound and protected from DNAase digestion by a promoter-specific transcription factor, Adf-1. Analysis of proximal promoter mutants identified multiple upstream elements that influence transcription, and DNAase footprint analysis detected three specific binding regions. Adf-1 binds at least one of these proximal promoter regions but interaction at this site is not specifically required for transcription. Our results suggest that multiple sequence-specific DNA binding proteins interact differentially with the proximal and distal promoters of Adh to activate transcription.