We have hypothesized that the co-localization of digestive zymogens with lysosomal hydrolases, which occurs during the early stages of every experimental pancreatitis model, facilitates activation of those zymogens by lysosomal hydrolases such as cathepsin B and that this activation triggers acute pancreatitis by leading to acinar cell injury. Some, however, have argued that the co-localization phenomenon may be the result, rather than the cause, of zymogen activation during pancreatitis. To resolve this controversy and explore the causal relationships between zymogen activation and other early pancreatitis events, we induced pancreatitis in mice by repeated supramaximal secretagogue stimulation with caerulein. Some animals were pre-treated with the cathepsin B inhibitor CA-074me to inhibit cathepsin B, prevent intrapancreatic activation of digestive zymogens, and reduce the severity of pancreatitis. We show that inhibition of cathepsin B by pre-treatment with CA-074me prevents intrapancreatic zymogen activation and reduces organellar fragility but it does not alter the caerulein-induced co-localization phenomenon or sub-cellular F-actin redistribution or prevent caerulein-induced activation of NF-B, ERK 1/2, and JNK, or up-regulated expression of cyto-chemokines. We conclude (a) that the co-localization phenomenon, F-actin redistribution, activation of pro-inflammatory transcription factors, and up-regulated expression of cyto-chemokines are not the result of zymogen activation, and (b) that these early events in pancreatitis are not dependent upon cathepsin B activity. In contrast, zymogen activation and increased sub-cellular organellar fragility during caerulein-induced pancreatitis are dependent upon cathepsin B activity.