We have previously demonstrated that activation of the Na(+)-Ca2+ exchanger in the reverse mode causes Ca2+ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na(+)-Ca2+ exchanger is involved in agonist-induced Ca2+ signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by L-glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca2+]i. The NA-induced Ca2+ signal was also attenuated by S-nitroso-L-cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na(+)-Ca2+ exchanger enhanced the ionomycin-induced increase in [Ca2+]i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca2+ signal. Furthermore, the ionomycin-induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na(+)-Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and that the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes.