The major physiological function of exocrine acinar cells from the pancreas and salivary gland is the secretion of proteins and fluid which are initiated by changes in cytosolic Ca²⁺ following neurotransmitter or hormone exposure. It is established that the spatiotemporal characteristics of the Ca²⁺ signal are vitally important for the appropriate stimulation of secretion and these properties are often disrupted in disease states. Experiments performed in isolated tissue have documented the complexity of these signals including sub-cellularly restricted signals, intra and inter-cellular Ca²⁺ waves, and apparent pacing of signals within individual acinar clusters by initiator cells. Whether these characteristics are mirrored in vivo was not known. To address this question, we have generated mice expressing the genetically encoded Ca²⁺ indicator GCamp6f specifically in acinar cells and developed an imaging platform to study the characteristics of Ca²⁺ signals in vivo in anesthetized mice by multi-photon microscopy. In submandibular salivary acinar glands (SMG), we show that stimulation of intrinsic nerves to the gland result in rapid oscillatory Ca²⁺ signals following ACh release. These events are positively correlated with fluid secretion. These signals appear to initiate in specific cells within individual acini and propagate to neighboring cells. In pancreas, Ca²⁺ signals were observed following neural stimulation that were dependent on ACh release. Ca²⁺ signals as a function of elevated serum cholecystokinin were observed in fasted animals that were augmented in terms of the number of responding cells and peak response following feeding. Both nerve stimulation and CCK induced Ca²⁺ oscillations in pancreatic acini, but with markedly distinct temporal and spatial characteristics. We speculate that initiating cells in each gland are more directly stimulated, either by direct neural innervation or by proximity to the vasculature or alternatively represent cells most sensitive to secretagogue by virtue of receptor number. In total, these studies define the physiological characteristics of Ca²⁺ signals in vivo and the platform will be useful in future investigation of disruption of Ca²⁺ signaling in disease states of exocrine tissue.