We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)alpha. PKCalpha subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKC(&agr;) activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCalpha, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKC(&agr;) activation arrested cells in the G(1) phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21(cip1) cyclin-dependent kinase inhibitors. Levels of p21(cip1) remained elevated and CDK2 activity repressed in spite of PKCalpha downregulation, indicating that downstream effectors of PKCalpha are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCalpha-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCalpha results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.