The activation of muscarinic receptors in N1E-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value approximately 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in approximately 10 s after washing the agonist away. The current density is 0.11 +/- 0.08 pA/pF (mean +/- SD; n = 12). It is absent in zero-Ca++ saline and reduced by Mn++ and Ba++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 +/- 41 nM (n = 7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 +/- 0.20 pA/pF (n = 6). Thapsigargin activated current is reduced by Mn++ and Ba++ and increased by elevated external Ca++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 +/- 35 nM. The Ca++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R. Penner. 1993. Journal of Physiology. 465:359-386; Zweifach, A., and R. S. Lewis, 1993. Proceedings of the National Academy of Sciences, USA. 90:6295-6299).