Prior stress ameliorates caerulein-induced pancreatitis in rats. NF-κB is a proinflammatory transcription factor activated during caerulein pancreatitis. However, the effects of prior stress on pancreatic NF-κB activation are unknown. In the current study, the effect of prior water immersion stress on caerulein and tumor necrosis factor-α (TNF-α)-induced NF-κB activation in the pancreas was evaluated. Water immersion of rats for up to 6 h prevents supramaximal caerulein-induced pancreatic IκB-α degradation and NF-κB activation in vivo. NF-κB activity is also inhibited in vitro in pancreatic acini prepared from water-immersed animals. TNF-α-induced NF-κB activation in pancreas or in pancreatic acini is unaffected by prior water immersion. Chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate/acetoxymethyl ester has similar effects to water immersion in preventing caerulein but not TNF-α-induced NF-κB activation in pancreas. Both the spike response and the sustained rise in [Ca2+]i in response to supramaximal caerulein stimulation are reduced markedly in acini prepared from water-immersed animals as compared with normal animals. Our findings indicate that, in addition to Ca2+-dependent mechanisms, Ca 2+-independent signaling events also may lead to NF-κB activation in pancreatic acinar cells. Water immersion stress prevents supramaximal caerulein-induced NF-κB activation in pancreas in vivo and in vitro by affecting intracellular Ca2+ homeostasis.