Normal human colonic luminal ammonium (NH₄ ⁺) concentration ranges from approximately 10- 100 mM. Despite this, the nature of NH₄ ⁺ effects on transport as well as NH₄ ⁺ transport itself in colonic epithelium is poorly understood. We elucidate here the effects of apical NH₄ ⁺ on cAMP-stimulated Cl^- secretion in colonic T84 cells. In HEPES buffered solutions apical NH₄ ⁺ at 10 mM had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH₄ ⁺ reduced current within 5 min to 61%±4% in the presence of 25 mM HCO₃ ^-. Current inhibition was not simply due to an increase in extracellular K ⁺ like cations in that the current magnitude was 95±5% with apical 10 mM K ⁺, and 46±3% with 10 mM NH₄ ⁺, relative to apical 5 mM K ⁺. We have previously demonstrated that inhibition of Cl^- secretion by basolateral NH₄ ⁺ occurs in HCO₃ ^- - free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH₄ ⁺ inhibition of current in HCO₃ ^- - buffer did not show anomalous mole fraction behavior, and followed the absolute [NH₄ ⁺] in K ⁺/NH₄ ⁺ mixtures where [K ⁺]+[NH₄ ⁺] = 10 mM. The apical NH₄ ⁺-inhibitory effect was not prevented by 100 µM Methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH₄ ⁺ inhibition of current was prevented by 10 min pretreatment with apical 500 µM DIDS, 100 µM DNDS, or 25 µM niflumic acid thus suggesting a role for NH₄ ⁺ action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 (DRA) and SLC26A6 (PAT1) were detected in T84 cells by RT-PCR, northern and western blots. Both DRA and PAT1 appear to co-associate with CFTR in the apical membrane. We conclude that the HCO₃ ^--dependence of apical NH₄ ⁺ inhibition of secretion is due to the action of NH₄ ⁺ on an apical anion exchanger. Supported by NIH DK051630.