Transforming growth factor β (TGF-β) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-β. TGF-β overexpression inhibits tumorigenesis, and abolition of TGF-β signaling accelerates tumorigenesis, suggesting that TGF-β acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-β-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-β to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-β to inhibit the proliferation of E2F1-, c-Myc-, and ^V12H-Ras-transformed cells, even though these cells were insensitive to TGF-β-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-β-induced growth arrest could not be explained by increases in TGF-β receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-β type I receptor. Significantly, TGF-β and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-β-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr¹⁶⁰. Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-β cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.