Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes "capacitative calcium entry" in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance.