N-Acetylcysteine (NAC) has been widely used as an antioxidant in research, however, it has also been found to reduce the binding of TNF to its receptor independent of its antioxidative role. In this study, we investigated the effect of NAC on NF-kappaB activation. In HeLa cells, Hep3B cells, and A549 cells, DNA-binding activity of NF-kappaB was induced by NAC without any other stimulation but not by tetramethylthiourea (TMTU) or vitamin C, suggesting that ROS is not involved in the effect of NAC. The degradation of IkappaBalpha and nuclear translocation of NF-kappaB were not induced by NAC. The phosphorylation of p65 at serine 536 was induced by NAC, which is known to contribute to the enhancement of DNA-binding activity of NF-kappaB, however, NAC did not directly phosphorylate p65. The NAC-induced DNA-binding activity of NF-kappaB and phosphorylation of p65 were sensitive to a phosphatidylinositol (PI) 3-kinase inhibitor, partially sensitive to an IkappaB kinase (IKK) inhibitor, but not sensitive to a Bruton's tyrosine kinase (Btk) inhibitor. Moreover, both the DNA-binding activity and phosphorylation induced by NAC were reduced by the overexpression of a dominant negative Akt in HeLa cells. These results suggest that NAC activates mainly PI3K to phosphorylate p65 and subsequently induces DNA-binding activity of NF-kappaB, independent of its antioxidative function.