We use chromatin immunoprecipitation to show that genes on the two active X chromosomes in undifferentiated, XX female embryonic stem cells (ES cells) are marked by hyperacetylation of all core histones, hyper(di)methylation of H3 lysine 4 and hypo(di)methylation of H3 lysine 9, compared with autosomal genes or genes on the single active X in XY male cells. The mark is found on both coding and promoter regions. On differentiation, and after the onset of X inactivation, the mark is reversed on the inactive X, whose genes show extreme hypoacetylation of all four core histones, hypo(di)methylation of H3K4 and hyper(di)methylation of H3K9. The mark is retained on the active X in female ES cells for at least several days of differentiation, but is not present in adult females. The selective marking of X-linked genes in female ES cells in a way that distinguishes them from the equivalent genes in males, is unprecedented. We suggest that the mark forms part of a chromatin-based mechanism that restricts X-inactivation to cells with more than one X chromosome.