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Using chromatography on a Fast S-Sepharose column, the insulin-stimulated S6 kinase can be resolved from other S6 kinases present in 3T3 L1 cell extracts. Only one S6 kinase is greatly activated by insulin (4–5-fold) and phosphorylates S6 with a high stoichiometry (4–5 mol phosphate per mol S6). This insulin-dependent S6 kinase can be activated in cell-free extracts by incubation with high concentrations of Ca2+. This activation is blocked by protease inhibitors such as leupeptin and is mimicked by trypsin. The stimulation does not require the presence of the protein kinase dependent on phospholipids and calcium (PK-C) in the cell extracts. The level of stimulation produced by proteolysis in the cell extracts is comparable to that reached in vivo by incubation with insulin.