Protein kinase C (PKC) isoforms are involved in the transduction of a number of signals important for the regulation of cell growth, differentiation, apoptosis and other cellular functions. PKC proteins reside in the cytoplasm in an inactive state translocate to various membranes to become fully activated in the presence of specific cofactors. Recent evidence indicates that PKC isoforms have an important role in the nucleus. We recently showed that insulin rapidly increases PKC RNA and protein. In this study we initially found that insulin induces an increase in PKC protein in the nuclear fraction. We therefore attempted to elucidate the mechanism of the insulin-induced increase in nuclear PKC. Studies were performed on L6 skeletal myoblasts and myotubes. The increase in nuclear PKC appeared to be unique to insulin, as it was not induced by other growth factors or Rosiglitazone. Inhibition of transcription or translation blocked the insulin-induced increase in nuclear PKC, whereas inhibition of protein import did not. Inhibition of protein export from the nucleus reduced the insulin-induced increase in PKC in the cytoplasm, and increased it in the nucleus. The increase in nuclear PKC induced by insulin was reduced but not abrogated by treatment of isolated nuclei by trypsin digestion. Finally, we showed that insulin induced incorporation of ³⁵S-methionine into nuclear PKC protein; this effect was not blocked by inhibition of nuclear import. Thus, these results suggest that insulin may induce nuclear-associated, or possibly nuclear, translation of PKC protein.