Post-translational modifications of histone amino termini are an important regulatory mechanism that induce transitions in chromatin structure, thereby contributing to epigenetic gene control and the assembly of specialized chromosomal subdomains 1, 2. Methylation of histone H3 at lysine 9 (H3–Lys9) by site-specific histone methyltransferases (Suv39h HMTases) 3 marks constitutive heterochromatin 4, 5, 6. Here, we show that H3–Lys9 methylation also occurs in facultative heterochromatin of the inactive X chromosome (X i) in female mammals. H3–Lys9 methylation is retained through mitosis, indicating that it might provide an epigenetic imprint for the maintenance of the inactive state. Disruption of the two mouse Suv39h HMTases abolishes H3-Lys9 methylation of constitutive heterochromatin but not that of the X i. In addition, HP1 proteins, which normally associate with heterochromatin, do not accumulate with the X i. These observations suggest the existence of an Suv39h-HP1-independent pathway regulating H3-Lys9 methylation of facultative heterochromatin.