The cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1 is a multidomain, multifunctional protein and a candidate tumor suppressor. Here, we show that, among rationally designed and tumor-associated mutants of human p21 ectopically expressed in U-2-OS cells, those that are selectively deficient in binding to either cyclin or CDK are partially impaired in inhibiting endogenous CDK activities but efficiently promote assembly of active cyclin D/CDK4(6) complexes. These results provide mechanistic insights into the p21-cyclin/CDK interplay in vivo and suggest a functional subclassification of tumor-specific aberrations of p21. Intriguingly, the subclass exemplified by the melanoma-derived N50S mutant may promote tumorigenesis, by both attenuating CDK-inhibitory function and concomitantly activating the proto-oncogenic cyclin D-dependent kinases.