Ca2+ extrusion was measured simultaneously with the free intracellular Ca2+ concentration ([Ca2+]i) from single pancreatic acinar cells placed in microdroplets of extracellular solution (Tepikin, A. V., Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572). Submaximal stimulation with cholecystokinin usually evoked discrete cytosolic Ca2+ spikes and each of these spikes was associated with a discrete and virtually synchronous pulse of Ca2+ extrusion into the extracellular microdroplet solution. When ACh evoked repetitive discrete [Ca2+]i spikes, each spike was also accompanied by a discrete pulse of Ca2+ extrusion. The velocity of Ca2+ extrusion oscillated with a time course similar to that of [Ca2+]i. The extracellular solution in our experiments had a low total calcium concentration (15-35 microM) and only a limited number of [Ca2+]i spikes (2-8) could be evoked. The magnitudes of the [Ca2+]i spikes and the amounts of Ca2+ extruded during each spike gradually decreased in each experiment. During the first cholecystokinin-evoked cytosolic Ca2+ spike the Ca2+ extrusion corresponded to a loss of 15-70% (mean value 39% +/- 12) of the mobilizable cellular calcium pool. The substantial pulsatile Ca2+ extrusion occurring synchronously with the receptor-activated cytosolic Ca2+ spikes is therefore an important element in repetitively bringing back [Ca2+]i to the resting level.