In fura 2-loaded N1E-115 cells, regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) following a Ca²⁺ load induced by 1 µM thapsigargin and 10 µM carbonylcyanide p-trifluoromethyoxyphenylhydrazone (FCCP) was Na⁺ dependent and inhibited by 5 mM Ni²⁺. In cells with normal intracellular Na⁺ concentration ([Na⁺]_i), removal of bath Na⁺, which should result in reversal of Na⁺/Ca²⁺ exchange, did not increase [Ca²⁺]_i unless cell Ca²⁺ buffer capacity was reduced. When N1E-115 cells were Na⁺ loaded using 100 µM veratridine and 4 µg/ml scorpion venom, the rate of the reverse mode of the Na⁺/Ca²⁺ exchanger was apparently enhanced, since an ~4- to 6-fold increase in [Ca²⁺]_i occurred despite normal cell Ca²⁺ buffering. In SBFI-loaded cells, we were able to demonstrate forward operation of the Na⁺/Ca²⁺ exchanger (net efflux of Ca²⁺) by observing increases (~ 6 mM) in [Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could only be observed when a continuous ionomycin-induced influx of Ca²⁺ occurred. The voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to measure changes in membrane potential. Ionomycin (1 µM) depolarized N1E-115 cells (~25 mV). This depolarization was Na⁺ dependent and blocked by 5 mM Ni²⁺ and 250-500 µM benzamil. These data provide evidence for the presence of an electrogenic Na⁺/Ca²⁺ exchanger that is capable of regulating [Ca²⁺]_i after release of Ca²⁺ from cell stores.