Spontaneous secretions occur in both neurons and non-neuronal cells, and calcium is important for these secretion processes. However, the detailed roles of calcium on the secretions have not yet been identified. In the present study, cultured Xenopus myocytes loaded with exogenous acetylcholine (ACh) into the cytoplasm in the absence of extracellular Ca2+ undergo spontaneous quantal ACh secretion as detected by the appearance of pulsatile miniature endplate currents. Analysis of the frequencies, amplitudes, and time courses of these currents suggests that similar cellular mechanisms are involved in the secretions of ACh in normal medium and Ca2+-free solution. Various doses of ryanodine were used to regulate the intracellular Ca2+ to different levels. The spontaneous ACh secretion from myocytes in Ca2+-free medium was decreased by reducing intracellular Ca2+ levels and enhanced by increasing cytosolic Ca2+ levels. These observations demonstrate that the spontaneous secretion from isolated myocytes and the effect of ryanodine on ACh-loaded cells are both independent of extracellular Ca2+ while Ca2+ in the sarcoplasmic reticulum plays a crucial role in the secretions.