Activation of the tumor suppressor protein p53 contributes to cellular senescence. As glycogen synthase kinase-3 (GSK3) was recently found to interact with p53 and contribute to the actions of p53, this study examined whether GSK3 accumulated in the nucleus and associated with p53 in senescent cells. Compared with young and middle-aged human WI-38 fibroblasts, senescent cells were found to contain increased nuclear levels of GSK3beta, and also tended to accumulate in the nucleus the other isoform of GSK3, GSK3alpha. Co-immunoprecipitation experiments demonstrated that GSK3beta and p53 formed a complex in the nucleus. Further experiments tested whether inhibition of GSK3 altered the development of senescence using long-term treatment with the selective GSK3 inhibitor lithium. Lithium treatment reduced the senescence-associated accumulation of p53 and caused cells to enter a reversible quiescent state. These results indicate that a portion of the p53 that is activated in senescent cells is modulated by its association with GSK3beta in the nucleus, an association that is known to facilitate the actions of p53 and that may contribute to senescence.