The ribosomal protein S6 is essential for the formation of the subunits of higher eukaryotic ribosomes, and S6 heterozygosity leads to early embryonal lethality in mice. S6 is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. So far, the S6Kinases, S6K1 and S6K2 are the only proven S6 S240/244 phosphorylating enzymes in mammalian cells. The activity of these S6Kinases is strictly regulated via the mammalian target of rapamycin (mTOR) enzyme complex with raptor, named mTORC1. In time course experiments with the mTORC1 inhibitor rapamycin we here demonstrate rapamycin-resistant phosphorylation of the ribosomal protein S6 at S240/244. Serum-restimulation experiments further demonstrated that this rapamycin-resistant S6 240/244 phosphorylation is induced via serum factors in a cell cycle-dependent manner. Our data allow new insights into the regulation of S6 phosphorylation and provide evidence for the existence of rapamycin-resistant S6 phosphorylating kinase activities.