Serine 10 phosphorylation of histone H3 (H3S10ph) has long been considered a mitotic marker, which is often associated with chromosome condensation both in plants and animals. Yet, in animal cells, H3S10ph was found associated with transcriptional activation of genes. Here we extend this view to plant cells showing that H3S10ph not only occurs in dividing cells during mitosis, but also in differentiated mesophyll cells. In these cells H3S10ph displayed a peculiar localization within the nucleolus where it was restricted to specific domains reminiscent of fibrillar centers. Chromatin immunoprecipitation analysis showed that H3S10ph is associated with ribosomal DNAs. Thus, in plants H3S10ph appears to be associated with two structurally differing nuclear sites engaged in gene silencing (mitotic centromeres) and in gene transcription (nucleolus).