In salivary acinar cells, the pattern of the Ca2+ signals that regulates fluid and enzyme secretion has yet to be resolved, as there are conflicting reports in the literature. We have used a two-photon technique to directly visualize the acinar cell lumen in living fragments of exocrine tissue and simultaneously recorded agonist-induced changes in intracellular Ca2+. We show near-synchronous global Ca2+ responses in submandibular acinar cells, distinct from the typical apical to basal Ca2+ wave usually seen in rodent pancreatic acinar cells. In an effort to explain the basis of these near-synchronous global Ca2+ responses we used immunocytochemical experiments to localize luminal proteins and inositol trisphosphate receptors (InsP3Rs) in tissue fragments. Zona occludens 1 (ZO-1), a tight junction protein, shows that individual submandibular acinar cells are often nearly completely encircled by a narrow luminal structure. By contrast, in pancreatic fragments, ZO-1 staining shows short luminal branches terminating abruptly at the apical pole of single acinar cells. Co-immunostaining of InsP3Rs type 2 and type 3 showed them in the same region as ZO-1 in both exocrine tissues. Functional experiments showed that the near-synchronous global Ca2+ responses were still observed in the absence of extracellular Ca2+ and also in the presence of ryanodine. We conclude that the elaborate luminal region of submandibular cells leads to a hitherto unrecognized extensive distribution of InsP3Rs in a band around the cell and that this underlies the near-synchronous global Ca2+ response to agonists. We suggest that this may be a structural adaptation in submandibular cells to support the copious amounts of fluid secreted.