Inflammation and cell death are critical to pathogenesis of acute pancreatitis. Here we show that transcription factor nuclear factor-κB (NF-κB), which regulates these processes, is activated and plays a role in rat cerulein pancreatitis. NF-κB was strongly activated in the pancreas within 30 min of cerulein infusion; a second phase of NF-κB activation was prominent at 3–6 h. This biphasic kinetics could result from observed transient degradation of the inhibitory protein IκBα and slower but sustained degradation of IκBβ. The hormone also caused NF-κB translocation and IκB degradation in vitro in dispersed pancreatic acini. Both p65/p50 and p50/p50, but not c-Rel, NF-κB complexes were manifest in pancreatitis and in isolated acini. Coinfusion of CCK JMV-180, which abolishes pancreatitis, prevented cerulein-induced NF-κB activation. The second but not early phase of NF-κB activation was inhibited by a neutralizing tumor necrosis factor-α antibody. AntioxidantN-acetylcysteine (NAC) blocked NF-κB activation and significantly improved parameters of pancreatitis. In particular, NAC inhibited intrapancreatic trypsin activation and mRNA expression of cytokines interleukin-6 and KC, which were dramatically induced by cerulein. The results suggest that NF-κB activation is an important early event that may contribute to inflammatory and cell death responses in acute pancreatitis.