Problem The mTOR pathway, a central regulator of cell metabolism, proliferation, and survival, is activated in a 100% of HNSCC. We have previously shown the mTOR inhibitor CCI-779 (temsirolimus; rapamycin analogue) inhibits growth of FaDu HNSCC in vitro and in vivo and significantly reduced microvessel density in HNSCC xenografts. Hence to study stromal effects of temsirolimus, FaDu cells were stably transfected with rapamycin resistant mTOR (rr-mTOR) to elucidate anti-tumor mechanisms of temsirolimus. Methods FaDu cells stably transfected with control vector or rr-mTOR. HNSCC tumor xenografts were established in nude mice by subcutaneous injection of control vector transfected (cv-FaDu) or rr-mTOR transfected (rr-FaDu) cells. Temsirolimus (5 mg/kg) was evaluated in mice treated for 3 weeks. PCR analysis of the 5'-end of an exogenous rr-mTOR sequence was performed on DNA isolated from explanted tumor tissues. Results Growth of cv-FaDu was suppressed by rapamycin (100 ng/ml), while the rr-FaDu clone was insensitive. PCR assay yielded the expected 246 b.p. product in rr-FaDu, but not in cv-FaDu xenografts indicating the presence of the exogenous rr-mTOR coding sequence in rr-FaDu xenograft tumor tissues. Temsirolimus inhibited growth of cv-FaDu and rr-FaDu xenograft tumors and was comparable in both groups suggesting the ‘anti-tumor’ activity of the drug in vivo is predominantly a consequence of its anti-stromal effects. Conclusion Results of this study suggest tumor growth inhibitory effects of temsirolimus on HNSCC xenografts are mediated mainly by the anti-stromal activity of the drug rather than by direct anti-tumor effect alone. Significance Understanding the mechanism of action of mTOR inhibition is important in the management of HNSCC as the Akt/mTOR pathway is activated in 100% of HNSCC. Support This work was supported by NCI grant R01CA102363 to Cherie-Ann O. Nathan.