S100A6 (calcyclin) is a calcium-binding protein of cell-specific expression whose gene is clustered with other S100 genes within the epidermal differentiation complex, on human chromosome 1q21. Many S100 proteins, including S100A6, are expressed in human epidermis at various stages of differentiation and their expression is often deregulated in skin and epithelial cancers. To gain insight into the mechanism of regulation of S100A6 expression, we examined epigenetic marks, that is DNA methylation and histone modifications along the S100A6 gene. Sequencing of bisulfite-modified DNA within a 3,247 bp long genomic region encompassing the promoter/first exon CpG island, the coding sequence of the S100A6 gene and a downstream region showed that it is almost entirely methylation-free in S100A6 expressing human epidermoid carcinoma (Hep-2) cells and lymphocytes and methylated in S100A6-negative embryonic epithelial (HEK293) cells. Chromatin immunoprecipitation revealed profound differences in the level of histone H3 acetylation and methylation and in the in vivo binding of upstream regulatory factor (USF), to the S100A6 gene promoter in S100A6-negative and -positive cells. These data demonstrate that cell-specific S100A6 expression is under control of epigenetic mechanisms.