Human p21 (also known as WAF1, CIP1, or SDI1) is a dual inhibitor of cyclin dependent kinases (CDKs) and the replication factor PCNA, which plays a role as a downstream mediator of the cell-cycle arrest induced by the tumor suppressor p53. To determine whether inactivation of downstream targets of p53 might contribute to cellular transformation, we have examined the integrity of the p21 gene in 36 invasive ductal breast carcinomas. Direct sequence analysis of the polymerase chain reaction-amplified p21 gene revealed a C to T transition in codon 94 that caused the substitution of a tryptophan for an arginine in a tumor specimen. This mutation was not detected in normal DNA extracted from the same patient nor in a polymerase chain reaction-restriction fragment length polymorphism of 50 unrelated individuals, indicating that it corresponds to a tumor-specific alteration. Functional analysis of the p21(R94W) protein produced in different eukaryotic and prokaryotic expression systems revealed that this mutation impaired the ability of p21 to inhibit CDKs. By contrast, the R94W mutant was unaltered in its ability to promote cyclin-CDK association as well as in its ability to bind proliferating cell nuclear antigen, thus leaving its putative functions as kinase activator or as inhibitor of replicative DNA synthesis intact. On the basis of these functional analysis, we propose that the Arg residue at position 94 is important for the CDK inhibitory role of p21.