In normal cells, HIF1a is the critical isoform regulating the response to hypoxia. In clear cell RCC, HIF2a appears to drive tumor progression. Therefore, the inhibition of both mTORC1 and mTORC2 has the potential to be highly effective for inhibiting clear cell RCC. Consistent with this possibility, we found that clinical renal tumors had increased expression of genes associated with mTOR activity that were both sensitive and insensitive to mTORC1 inhibition. Cho et al reported that a second generation mTOR inhibitor ASP1517 HIF inhibitor targeting mTOR and PI3 Kinase decreased the level of HIF2a, while rapamycin did not. Ku0063794 is a second generation mTOR inhibitor targeting mTORC1 and mTORC2. Ku0063794 was compared with temsirolimus using preclinical LY2157299 models of RCC. The 786-O cells are VHL2/2 and have constitutive HIF activity while Caki-1 cells are VHL +/+. These are two widely used human RCC lines that are documented to be derived from the clear cell variant of RCC. Table S1 summarizes the results of cell signaling studies. In human RCC cell lines, Ku0063794 inhibited the activity of both mTORC1 and mTORC2, while temsirolimus activity was generally limited to mTORC1. Our study suggests that phosphorylation of mTOR at Ser2448 and Ser2481 is primary regulated by mTORC2 since phosphorylation was strongly inhibition by Ku0063794 but not temsirolimus. However, prior reports do not firmly assign these phosphorylation sites to mTORC2. Our results also suggest that Ser2448 and Ser2481 of mTOR may not accurately reflect either mTORC1 or mTORC2 activity since phosphorylation of targets downstream of mTOR preceded phosphorylation of Ser2448 and Ser2481. In our study, temsirolimus produced a transient decrease in the phosphorylation of AKT on Ser473 and Thr308, which are considered mTORC2 phosphorylation sites. This suggests that temsirolimus has some direct or indirect effect on this particular mTORC2-regulated phosphorylation. The effect may be brief because mTORC1 inhibition removes negative feedback loops targeting AKT; and increased AKT activity quickly overcomes any minor mTORC2 inhibition provided by temsirolimus. In vitro cell viability studies were used to assess the direct effect of Ku0063794 and temsirolimus on human RCC cell lines. Ku0063794 decreased the viability of RCC cell lines in both a concentration and time dependent manner. In contrast, increasing the concentration of temsirolimus had a relatively small effect on cell viability, even though the concentrations tested included pharmacologically relevant concentrations.