In non-excitable cells, like exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca²⁺ signals consist of both Ca²⁺ release and Ca²⁺ influx. We have investigated the contribution of these processes to membrane localized Ca²⁺ signals in pancreatic and parotid acinar cells using Total Internal Reflection Fluorescence (TIRFM) microscopy. This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and cover-slip. In TIRFM mode, physiological agonist stimulation resulted in Ca²⁺ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional Wide-Field Microscopy (WFM). Because local Ca²⁺ release in the TIRF zone would be expected to saturate the Ca²⁺ indicator (Fluo-4), these data suggest that Ca²⁺ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca²⁺ release, followed by a substantial, maintained plateau phase indicative of Ca²⁺ entry, was observed. The contribution of Ca²⁺ influx and Ca²⁺ release in isolation to these near plasma membrane Ca²⁺ signals was investigated by using a Ca²⁺ readmission protocol. In the absence of extracellular Ca²⁺, the profile and magnitude of the initial Ca²⁺ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition was similar to that obtained with WFM in both pancreas and parotid acini. In contrast, when Ca²⁺ influx was isolated by subsequent Ca²⁺ readmission, the Ca²⁺ signals evoked were more robust than measured with WFM. Furthermore, in parotid acinar cells, Ca²⁺ readdition often resulted in the apparent saturation of Fluo-4, but not of the low affinity dye Fluo-4-FF. Interestingly, Ca²⁺ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca²⁺ influx was observed in parotid acinar cells even at low, physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near membrane Ca²⁺ signals mediated by Ca²⁺ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.