The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.