Enzyme secretion in pancreatic acinar cells is dependent on the exocytosis of secretory granules (1). After fusion with plasma membrane the granules remain close to the plasma membrane for many minutes and, at least in the early stages after fusion, the fusion pore is open (2). In other systems fusion pore dynamics have been observed and shown to be a factor in the release of granule contents (3). However, any dynamic to the behaviour of the fusion pore in acinar cells has not been studied. In the present study we have set out to determine if fusion pore dynamics are present in pancreatic acinar cells. Fragments of mouse exocrine pancreas were prepared by a collagenase digestion method. In control experiments Lysine-fixable Texas Red (TR) and Fluorescein dextran (FD) were added to the extracellular solution before cell stimulation. These extracellular dyes then entered and labelled granules that had undergone exocytosis stimulated with ACh. The tissue fragments were then fixed in 4% PFA and the staining visualized with a confocal microscope. In experiments designed to study fusion pore dynamics, TR and FD application were separated in time. TR was added at the beginning of all experiments and, to probe for fusion pore closure, FD was added 2, 6 or 11 min after cell stimulation with ACh. To measure fusion pore dynamics in live cells we used 2-photon microscopy and visualised fusion pore dynamics by the simultaneous imaging of extracellular dyes sulforhodamine (SRB) and methoxypyrenetrisulfonic acid (MPTS). In control experiments the simultaneous application of FD and TR before cell stimulation produced, as expected, identical patterns of staining. The FD/TR fluorescence ratio, measured in each individual granule, had a mean value of 0.84 ± 0.28 (mean ± SEM, n=175 granules) with no granules showing ratios lower than 0.2 indicating that granules are filled approximately equally with both dyes. When FD was added at later time points the FD/TR ratio subtly changed. For example, application of FD 2 min after stimulation gave a mean FD/TR ratio of 0.78 ± 0.43 (mean ± SEM, n=113). A Shapiro-Wilk normality test showed the control FD/TR ratio distribution was not significantly different from a Gaussian (P=0.2) but when FD was added after a 2 min delay the distribution was significantly skewed (P<0.05) with a predominance of low FD/TR ratios. For example, now 12.38% of granules had a ratio of less than 0.2 These small ratios indicate that these granules were filled predominantly with TR dye and we conclude that the fusion pore must have closed preventing entry of FD. Experiments using the 2-photon technique were performed in a similar manner, with SRB present throughout the experiment and the MPTS added at later time points to probe for fusion pore closure. We then imaged the granules labelled with SRB only (indicative of fusion pore closure) and after a period of time were able to observe sudden filling of the granule with MPTS, indicating fusion pore reopening. In conclusion, we show the fusion pore in acinar cells is dynamic. We hypothesize that this behaviour may be important in the regulation of enzyme release.