Induction of transglutaminase 2 (TGase 2) by epidermal growth factor (EGF) in human breast cancer cells increases their oncogenic potential and chemoresistance. The role of TGase 2 in the development of these tumor-related phenotypes remains to be elucidated, but it has been shown that expression of a dominant-negative form of TGase 2 reverses EGF-mediated chemoresistance in breast cancer cells. We examined several different breast cancer cell lines, representing both EGF receptor (EGFR)-positive and EGFR-negative breast cancers, and found that doxorubicin-resistant cells had a higher level of TGase 2 compared with doxorubicin-sensitive cells independent of the EGFR expression level. TGase 2 inhibition increased the chemosensitivity of drug-resistant cells, concomitant with a decrease in nuclear factor-kappaB (NF-kappaB) activity. Increasing the level of TGase 2 in drug-sensitive cells by transient transfection reduced the level of inhibitory subunit alpha of NF-kappaB (IkappaBalpha) and increased NF-kappaB activity in these cells. Inhibition of TGase 2 in drug-resistant cells by RNA interference increased the levels of IkappaBalpha, and this correlated with a shift in the accumulation of NF-kappaB from the nucleus to the cytosol. We recently showed that TGase 2 activated NF-kappaB through polymerization and depletion of free IkappaBalpha during inflammation. Therefore, increased expression of TGase 2 and subsequent activation of NF-kappaB may contribute to drug resistance in breast cancer cells independently of EGF signaling.