The effect of nitric oxide (NO) on Na+/H+exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10−3M S-nitroso- N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a ∼45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP ( P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1 H-(1,2,4)oxadiazolo(4,3- a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 μM); 4) chelerythrine chloride (2 μM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 μM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.