In normal human fibroblast cells, the primary cell cycle regulators, the cyclin-dependent kinases (CDKs), exist predominantly in multiple quaternary complexes, each consisting of a CDK, a cyclin, proliferating cell nuclear antigen (PCNA) and p21. p21 encodes a universal inhibitor of cyclin-dependent kinases. Here we show that the level of p21 mRNA and the interaction of p21 protein with cyclin-CDK enzymes are regulated during the cell cycle. When normal human fibroblast IMR90 cells were released from serum starvation, p21 mRNA reached its highest level immediately following serum stimulation, began to decrease at the G1/S boundary, fell to its lowest level during S phase, and accumulated again as cells exited from S phase. p21 protein associates with each cyclin-CDK complex in a cell cycle dependent manner. Cyclin A-CDK2-p21-PCNA and Cyclin B1-CDC2-p21-PCNA complexes are assembled in early S and G2 phase, respectively, indicating that p21 and/or PCNA regulates the enzymatic activity of each kinase at the time of their functioning. Cyclin D1-CDK4-p21-PCNA complexes, on the other hand, persist throughout the cell cycle, suggesting that cyclin D1-CDK4 quaternary complexes may play a role in monitoring an event(s) that may occur at any time, rather than at a specific stage of the cell cycle. The level of p21 mRNA in early passage Li-Fraumeni cells that are heterozygous for p53 mutation remained similar to that in normal fibroblasts, but was undetectable in immortalized Li-Fraumeni cells homozygous for mutant p53. This finding provides a plausible molecular explanation for the loss of genetic stability associated with cells homozygous, but not heterozygous, for p53 mutation.