Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-kappaB, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-kappaB, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKK alpha/beta, phosphorylation and degradation of IkappaB alpha, and phosphorylation of the p65 NF-kappaB subunit on serine 536. Proteasome inhibitor-induced NF-kappaB activity can be blocked by a non-degradable form of IkappaB alpha or dominant negative forms of either IKK alpha or IKK beta. Lentiviral delivery of shRNAs to either IKK alpha or IKK beta cause blockade of NF-kappaB transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IkappaB alpha degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-kappaB activation. Accordingly, siRNA knockdown of p65 inhibits proteasome inhibitor-induced NF-kappaB transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-kappaB instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NF-kappaB activation should be carefully examined in particular cell types.