The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso-N-acetyl pencillamine (SNAP) on Cl/OH exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl/OH exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 µM) for 30 min resulted in ~50% inhibition of DIDS-sensitive ³⁶Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl/OH exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl/OH exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl/OH exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl/OH exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl/OH exchange activity; 7) SO/OH exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of Cl/OH exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.