The human alcohol dehydrogenase gene ADH2 is expressed at high levels in the liver. At least five different complexes between nuclear proteins from mouse liver or rat hepatoma cells and the proximal promoter region extending from nucleotides -188 to +31 are detected by gel retardation assays. Using oligonucleotides to compete for the binding, and also as the probes, allowed localization of sequences within this region that bind nuclear proteins. Mutated oligonucleotides did not bind or compete. Nucleotides which are contacted by the proteins have been localized by methylation interference assays to two regions homologous to the related mouse Adh-1 gene. One maps between nucleotides -94 and -84; the other is from nucleotides -72 through -64. The 5' region of the human ADH2 gene is capable of directing accurate in vitro transcription in extracts from hepatoma cells. Deletion analysis indicates that the smallest portion of the proximal promoter region capable of directing significant transcription extends to nucleotide -93, which just contains both of the identified contact regions. The longer promoter fragments do not work as well, suggesting the presence of negative elements. In vitro transcription assays using oligonucleotides and mutated oligonucleotides as competitors demonstrate that both of the cis-acting sequences identified here are important to efficient initiation of transcription from the ADH2 promoter.