Following endocytic uptake of acetylcholine (ACh), CHO fibroblasts exhibit Ca(2+)-dependent spontaneous quantal ACh release and depolarization-evoked ACh release, as detected by a whole-cell voltage-clamped myocyte in contact with the fibroblast. CHO fibroblasts transfected with synaptotagmin I, an integral membrane protein of synaptic vesicles, showed a reduced spontaneous quantal ACh release and an enhanced Ca(2+)-evoked ACh release, as compared with control cells. Biochemical and ultrastructural studies of endocytic activity using horseradish peroxidase as a marker further confirmed the inhibitory action of synaptotagmin I on spontaneous vesicular exocytosis and on elevated exocytosis induced by Ca2+. Through inhibition of exocytosis at the resting intracellular concentration of Ca2+ and removal of the inhibition upon depolarization-induced Ca2+ entry, synaptotagmin I could enhance the efficiency of excitation-secretion coupling.