Tri-methylated lysine 20 on histone H4 (Me(3)K20H4) is a marker of constitutive heterochromatin in murine interphase and metaphase cells. Heterochromatin marked by Me(3)K20H4 replicates late during S phase of the cell cycle. Serum starvation increases the number of cells that exhibit high levels of Me(3)K20H4 at constitutive heterochromatin. Me(3)K20H4 is also present at the centromeric heterochromatin of most meiotic chromosomes during spermatogenesis and at the pseudoautosomal region, as well as at some telomeres. It is not present on the XY-body. During murine embryogenesis the maternal pronucleus contains Me(3)K20H4; Me(3)K20H4 is absent from the paternal pronucleus. On Drosophila polytene chromosomes Me(3)K20H4 is present in a 'punctate pattern' at many chromosomal bands, including the chromocenter. In coccids it is present on the facultatively heterochromatinised paternal chromosome set. We also present evidence that Me(3)K20H4 is dependent upon H3-specific Suv(3)9 histone methyltransferase activity, suggesting that there may be 'epigenetic cross-talk' between histones H3 and H4