The role of the Na⁺/Ca²⁺ exchanger in intracellular Ca²⁺ regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca²⁺-permeable glutamate receptors and L-type Ca²⁺ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca²⁺ stores regulate intracellular Ca²⁺ in these cells. We used the Ca²⁺-sensitive dye fluo 3 to measure changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) under conditions in which Na⁺/Ca²⁺ exchange was altered. In addition, the role of the Na⁺/Ca²⁺ exchanger in the refilling of the caffeine-sensitive Ca²⁺ store following caffeine-stimulated Ca²⁺ release was assessed. Brief applications of caffeine (1-10 s) produced rapid and transient changes in [Ca²⁺]_i. Repeated applications of caffeine produced smaller Ca²⁺ transients until no further Ca²⁺ was released. Store refilling occurred within 1-2 min and required extracellular Ca²⁺. Ouabain-induced increases in intracellular Na⁺ concentration ([Na⁺]_i) increased both basal free [Ca²⁺]_i and caffeine-stimulated Ca²⁺ release. Reduction of external Na⁺ concentration ([Na⁺]_o) further and reversibly increased [Ca²⁺]_i in ouabain-treated HC. This effect was not abolished by the Ca²⁺ channel blocker nifedipine, suggesting that increases in [Na⁺]_i promote net extracellular Ca²⁺ influx through a Na⁺/Ca²⁺ exchanger. Moreover, when [Na⁺]_o was replaced by Li⁺, caffeine did not stimulate release of Ca²⁺ from the caffeine-sensitive store after Ca²⁺ depletion. The Na⁺/Ca²⁺ exchanger inhibitor 2',4'-dimethylbenzamil significantly reduced the caffeine-evoked Ca²⁺ response 1 and 2 min after store depletion.