Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves that required gap junctional communication and influx of extracellular calcium through voltage-gated calcium channels. Gap junction-dependent intercellular calcium waves were inhibited by preventing plasma membrane depolarization. These studies demonstrate two distinct pathways by which insulin-secreting cells can coordinate cytosolic calcium rises, and show that it is by ionic traffic that gap junctions synchronize calcium-dependent events in these cells.