The Ca 2+‐and phospholipid‐binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca 2+‐affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 μM cytosolic free Ca 2+. The well‐characterized synaptotagmin isoforms I and II are present in pancreatic β‐cell lines RINm5F, INS‐1 and HIT‐T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin‐containing secretory granules whereas only minor amounts were found on synaptic vesicle‐like microvesicles. Antibodies or Fab‐fragments directed against the Ca 2+‐dependent phospholipid binding site of the first C 2 domain of synaptotagmin I or II inhibited Ca 2+‐stimulated, but not GTPγS‐induced exocytosis from streptolysin‐O‐permeabilized INS‐1 and HIT‐T15 cells. Transient expression of wild‐type synaptotagmin II did not alter exocytosis in HIT‐T15 cells. However, mutations in the Ca 2+‐dependent phospholipid binding site of the first C 2 domain (Δ180–183, D231S) again inhibited only Ca 2+‐, but not GTPγS‐evoked exocytosis. In contrast, mutations in the IP 4‐binding sites of the second C 2 domain (Δ325–341; K327, 328,332 Q) did not alter exocytosis. Synaptotagmin II mutated in both C 2 domains (Δ180–183/K327, 328,332 Q) induced greater inhibition than mutant Δ180–183, suggesting a discrete requirement for the second C 2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca 2+.