The overexpression of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1) can inhibit cell proliferation, which is mediated by direct binding to CDK and proliferating-cell nuclear antigen. In this study, we demonstrated that human cytosolic thymidine kinase 1 (TK1) polypeptide can form a complex with p21(Waf1). The C-terminal domain of p21(Waf1) appeared to interact with the TK1 polypeptide, but, despite the inhibitory function of p21(Waf1), their association did not alter TK1 functional activity. However, overexpression of TK1 overcame p21(Waf1)-mediated growth suppression and blocked the association of CDK2 with p21(Waf1), suggesting that TK1 interferes with the inhibitory function of p21(Waf1). Based on these results, we here propose that the molecular function of p21(Waf1) in cells can be perturbed through its interaction with another cellular protein, TK1.