Introduction: Pancreatic acinar cells overproduce cytokines following cell surface receptor hyperstimulation. Elucidation of mechanisms of acinar cell cytokine production is crucial for a better understanding of acute pancreatitis pathogenesis. Tumor necrosis factor-alpha (TNF) and interleukin-1-beta (IL-1) are key cytokines that initiate, maintain and propagate acute pancreatic inflammation. The role of stress kinases in acinar cell cytokine production is not completely understood. We hypothesize that the stress kinases p38 and ERK play an important role in acinar cell TNF and IL-1 production. Methods: Pancreatic fragments prepared from normal rats were equilibrated for 3 h in an incubator in tissue culture medium (5%CO2, 95%N, RT) and then treated with saline, or 100nM of the CCK-A receptor agonist caerulein, or 100nM caerulein and specific p38 inhibitor (10uM SB203580) or ERK inhibitor (100uM PD98059), or the inhibitor alone. After 3 h pancreatic fragments were homogenized and assayed for total and phosphorylated p38 and ERK, and for TNF and IL-1 concentrations (ELISA). Results (*ANOVA, p<0.05): Pancreatic fragments stimulated with caerulein showed several-fold increases* in TNF and IL-1 concentrations that were accompanied by significantly* increased phosphorylation of p38 and ERK. Specific p38 and ERK inhibitors significantly* inhibited caerulein-induced activation of the corresponding stress kinase and significantly* attenuated caerulein-induced IL-1 and TNF production by an impressive 50% margin. The increased activation of p38 and ERK in pancreatic fragments was not associated with significant increases in total-p38 and total-ERK concentrations, indicating that increases in p38 and ERK activation following caerulein-hyperstimulation are not a side-effect of stress kinase induction but are rather the consequence of phosphorylation of pre-existing p38 and ERK. The ERK inhibitor did not significantly inhibit p38 activation, and the p38 inhibitor did not significantly inhibit ERK activation, corroborating the specificity of the respective inhibitor. Furthermore, the inability of the p38 inhibitor SB203580 to inhibit activation of ERK following caerulein hyperstimulation does not support the recent view (J Biol Chem, June 3, 2005) that SB203580 may cross-react and antagonize the CCK-A receptor. In control groups, the individual stress kinase inhibitors used alone (without caerulein) showed no significant* effect. Conclusion: ERK and p38 play an important role in caerulein-stimulated acinar cell overproduction of IL-1 and TNF. (Support: American College of Surgeons Faculty Research Fellowship 2003-2005; NIH Career Development Award #K08-DK062805)