The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69+/-1.58% of the area, then the subplasmalemmal region with 12.61+/-0.77% and the perinuclear region with 9.07+/-0.97% ( n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9+/-1.95%, subplasmalemmal 12.45+/-0.78% and perinuclear regions 9.07+/-0.97% ( n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.