The pathologic activation of proteases within the pancreatic acinar cell is a key initiating event in acute pancreatitis. Past studies have suggested that the generation of a low-pH environment is critical to this process. Vacuolar adenosine triphosphatase (vATPase) is a multiprotein complex that transports protons across cellular membranes. Activation of the vATPase requires assembly of the soluble (V1) subunits on the membrane subunits (V0). It is found that conditions that cause protease activation in the acinar cell also cause assembly of V1 on V0. Further, inhibitors of vATPase block this protease activation. Ethanol and butanol sensitize the acinar cell to cholecystokinin-induced zymogen activation; vATPase inhibitors also blocked this activation. Activation of the vATPase may be central to the pathologic activation of proteases in the acinar cell and may also modulate the sensitizing effects of alcohols.