A new regulatory region (−108 to −86), named CE, containing potential CRE- and Ets-binding sites has been identified in the murine E-cadherin promoter. The Ets-binding site (at −97 position) negatively modulates the activity of the E-cadherin promoter in expressing keratinocyte cell lines and was responsible for the specific retarded complexes obtained with the CE region. Analysis of the methylation status of the endogenous E-cadherin promoter indicated that silencing of E-cadherin expression in malignant keratinocytes cannot be explained by hypermethylation mechanisms. Furthermore, treatment with 5′-aza-2′-deoxycytidine was unable to induce the expression of E-cadherin in deficient keratinocytes. However,in vivofootprinting analysis of the endogenous E-cadherin promoter showed a very distinct pattern in expressing and nonexpressing keratinocytes. Extensive interactions in the previously postulated proximal regulatory elements and in the CE region were detected in expressing cells, while only some nucleotides of the E-pal element and of the CE region were protected in nonexpressing keratinocytes. These results indicate a complex regulation of the mouse E-cadherin promoter and support a model where the combination of positive (CCAAT-box and GC-rich region) and negative (E-pal element and CE region)cis-acting elements contribute to the final level of E-cadherin gene expression. In addition, our results show that downregulation of E-cadherin expression in transformed epidermal keratinocytes is mainly exerted through the interaction of repressor factor(s) with the E-pal element and to the lack of interaction of positive acting factors with the proximal regions.