Premature activation of inactive digestive enzymes (or zymogens) within the pancreatic acinar cell is an initiating event in acute pancreatitis (AP). We have found that this response depends on the assembly and activation of an ATP-dependent proton pump, the vacuolar ATPase (vATPase). Previously, we have shown that the classic vATPase inhibitors concanamycin and bafilomycin can inhibit zymogen activation induced experimentally by high doses of the cholecystokinin orthologue, cerulein (CER) in isolated acinar cells. Recent studies have questioned the specificity of these inhibitors. In the current study we examine the role of the vATPase in pancreatitis using the newly developed novel vATPase inhibitors lobatomide-B and salicylihalamide-A as well as a genetic approach using siRNA. Both lobatomide-B and salicylihalamide-A inhibited CER stimulated zymogen (trypsinogen and chymotrypsinogen) activation but had no effect on amylase secretion. Lobatomide-B (0.1μM) was more potent, reducing activation to baseline levels. Treatment of cells with siRNA specific for the vATPase E-subunit (V1E) significantly decreased V1E expression. V1E siRNA also significantly decreased chymotrypsinogen activation, but not amylase secretion. These studies confirm a role for the vATPase in zymogen activation and demonstrate that the novel and specific inhibitors lobatomide-B and salicylihalamide-A reduce early pancreatitis responses.