Regulation of Adh-1 (encoding mouse alcohol dehydrogenase-A2) gene expression results from the interplay of positive and negative effects. We report here the detection and localization of a negative regulatory element (NRE) to the sequence between -754 and -732 bp relative to the transcription start point (tsp). This element, which we call Adh-1 NRE2, can down-regulate transcription from a strong heterologous promoter (the SV40 promoter+enhancer). Adh-1 NRE2 can function in both orientations and either upstream or downstream from the promoter, and thus has attributes characteristic of a 'silencer'. A protein that binds to the Adh-1 NRE is widespread in mouse tissues.