Under normal conditions, the expression of the murine type-I keratin K13 is restricted to the suprabasal, differentiating cell layers of internal stratified squamous epithelia that line the oral cavity and the upper digestive tract. It is, however, also expressed aberrantly but constitutively in only the differentiating parts of 7,12-dimethylbenz[alpha]anthracene/12.0-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) induced malignant epidermal tumors of the back skin of mice, whereas its likewise suprabasal expression in papillomas is highly variable [27]. In an approach to unravel regulatory DNA sequence elements involved in the tissue-specific and aberrant K13 expression, the 5'-flanking region of the gene was analyzed with regard to potential methylation sites and DNase hypersensitive regions. We report on the identification of a CpG dinucleotide (designated M1; located about 2.3 kb upstream of the transcriptional start site), whose methylation state correlates with the differential gene activity in various epithelia and tumors. We show that in K13-nonexpressing integumental epidermis the M1 site is methylated in both suprabasal and basal cells. In contrast, internal stratified squamous epithelia (i.e. tongue, esophagus, forestomach) exhibit an unmethylated M1 site not only in their suprabasal. K13-expressing cells, but also in basal cells--in which, however, the keratin is not yet synthesized. The identical situation is encountered in DMBA TPA-induced moderately differentiating epidermal squamous cell carcinomas with compartmentalized K13 expression. In papillomas we observed a striking correlation between the extent of both suprabasally expressed K13 protein and demonstrable DNA copies carrying an unmethylated M1 site. Moreover we found that the sequence region around the M1 site was DNAseI hypersensitive in K13-expressing malignant tumors, but DNaseI insensitive in K13-nonexpressing epithelia and cells. DNAseI hypersensitivity in K13-expressing tissues was, however, independent of an active transcription of the gene in differentiating cells or transcriptional inertia in basal cells. These results strongly suggest that the sequence element around the demethylated M1 site is involved in a multi-level control mechanism mediating the selective expression of the K13 gene in internal squamous epithelia and in DMBA/TPA-induced epidermal tumors.