Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance ( C m) measurements and by the membrane fluorescent dye FM1-43. When measured with the C mmethod, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3.5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s−1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of C m changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.