Objectives: To test the hypothesis that blockage of epidermal growth factor receptor (EGFR) or methylethylketone (MEK)1/2 kinase activities impairs the growth of kidney cancer cells and magnifies the growth inhibitory effect of the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Methods: The kidney cancer cells from eight cell lines (including a pair in which the VHL gene or an empty vector was transfected in a VHLmut cell line) were tested for the effect of treatment with an EGFR inhibitor or an MEK1/2 inhibitor on the phosphorylation status of the phosphatidyl inositol 3-OH kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways after stimulation with EGF. In vitro growth assays were performed with EGFR inhibitors (gefitinib or erlotinib) or with MEK1/2 inhibitors (UO126 or PD184352) alone or in combination with rapamycin. The effects of PD184352, rapamycin, and their combination in the cell cycle were evaluated by flow cytometry. Results: The growth suppressive effect of combined gefitinib (or erlotinib) and rapamycin was greater than the effect of each drug alone and was not dependent on VHL status. By affecting downstream signaling, the MEK1/2 inhibitors U0126 and PD184352 blocked growth more effectively than did the EGFR inhibitors in selected renal cell carcinoma lines; this effect was enhanced by the addition of rapamycin. At the cell cycle level, the combination resulted in enhanced G1 arrest. Although eIF4E overexpression has been suggested to make cells resistant to rapamycin, we observed marked growth inhibition with rapamycin as a single agent in SKRC39, which has marked overexpression of eIF4E. Conclusions: The results of our study have shown that combined mTOR and other upstream inhibitors have strong potential in the treatment of renal cell carcinoma. © 2007 Elsevier Inc. All rights reserved.