Ca2+ signaling and exocytosis are highly polarized functions of pancreatic acinar cells. The role of cellular architecture in these activities and the capacity of animals to tolerate aberrant acinar cell function are not known. A key regulator of acinar cell polarity is Mist1, a basic helix-loop-helix transcription factor. Ca2+ signaling and amylase release were examined in pancreatic acini of wild type and Mist1 null mice to gain insight into the importance of cellular architecture for Ca2+ signaling and regulated exocytosis. Mist1-/- acinar cells exhibited dramatically altered Ca2+ signaling with up-regulation of the cholecystokinin receptor but minimal effect upon expression of the M3 receptor. However, stimulation of inositol 1,4,5-trisphosphate production by cholecystokinin and carbachol was inefficient in Mist1-/- cells. Although agonist stimulation of Mist1-/- cells evoked a Ca2+ signal, often the Ca2+ increase was not in the form of typical Ca2+ oscillations but rather in the form of a peak/plateau-type response. Mist1-/- cells also displayed distorted apical-to-basal Ca2+ waves. The aberrant Ca2+ signaling was associated with mislocalization and reduced Ca2+ uptake by the mitochondria of stimulated Mist1-/- cells. Deletion of Mist1 also led to mislocalization of the Golgi apparatus and markedly reduced digestive enzyme content. The combination of aberrant Ca2+ signaling and reduced digestive enzyme content resulted in poor secretion of digestive enzymes. Yet, food consumption and growth of Mist1-/- mice were normal for at least 32 weeks. These findings reveal that Mist1 is critical to normal organelle localization in exocrine cells and highlight the critical importance of maintaining cellular architecture and polarized localization of cellular organelles in generating a propagating apical-to-basal Ca2+ wave. The studies also reveal the spare capacity of the exocrine pancreas that allows normal growth and development in the face of compromised exocrine pancreatic function.