The mouse alcohol dehydrogenase gene, Adh-1, is expressed in a tissue-specific manner. We examined the promoter activity of a series of 5' deletions extending from bp -473 to -47 and demonstrated that there are positive regulatory elements between bp -229 and +54 and a negative regulatory element between bp -323 and -229. To identify the sequence of the negative regulatory element, gel retardation and DNase I footprint assays were performed using nuclear proteins from mouse liver and from a hepatoma cell line, H4IIE-C3. A specific protein-binding site covered bp -324 and -297. Within this region, we identified sites of close protein-DNA contact by methylation interference assays, located in the sequence TGGAAGTTTCAGGTT (nt -316 to -302). Site-directed mutagenesis of four protein-DNA contact sites within this sequence eliminated the specific protein-DNA binding, assayed by gel retardation, and restored expression of Adh-1 in transient transfection assays to the levels seen when the entire region containing the negative element was deleted. Thus, we have identified a negative regulatory element within the Adh-1 promoter. No homology was found between this negative element and other regulatory elements, suggesting that this is a novel negative element.