Previous studies have shown conflicting data regarding cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes, and considering the widespread overexpression of cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown that cyclin D1 and Cdk2 form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with cyclin D1 and Cdk2 failing to complex in its absence. These p21/cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of cyclin D1/Cdk2 complexes, and that cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer, are shown to be phosphorylated by cyclin D1/Cdk2 complexes. Additionally, polypyrimidine tract binding protein-associated splicing factor (PSF) is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either cyclin E or cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity.