Higher eukaryotic ribosome biogenesis takes place in the nucleolus and requires the import of ribosomal proteins from the cytoplasm. The ribosomal protein S6 is essential for the formation of ribosome subunits, and in mice S6 heterozygosity triggers embryonal lethality. Downstream of the mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signalling pathways S6 protein is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. Here, we show that S240/244-phosphorylated S6 is predominantly nuclear but also detectable in the cytoplasm, whereas S235/236-phosphorylated S6 is almost exclusively localized to the nucleus of primary human cells and virtually undetectable in the cytoplasm. However, in transformed cells the latter can also be detected in the cytoplasm. Experiments with the mTOR inhibitor rapamycin revealed that neither blocking the phosphorylation of S6 at S235/236 and S240/244 nor arresting the cell cycle affects the cytoplasmic/nuclear localization of S6 protein. Our findings provide new insights into the regulation of S6 phosphorylation and S6 protein localization in mammalian cells.