A significant amount of NH₄⁺ is absorbed by the colon. The nature of NH₄⁺ effects on transport and NH₄⁺ transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of NH₄⁺ on cAMP-stimulated Cl^- secretion in the colonic cell line, T84. In HEPES buffered solutions application of basolateral NH₄⁺ resulted in a reduced level of Cl^- secretory current. The effect of NH₄⁺ appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K⁺, and 3) a long term (>20 min) increase in trans-epithelial resistance (TER). The competitive effect with K⁺ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K⁺ was inhibited 15% by 10 mM NH₄⁺ alone and by 30% with a mixture of 2 mM K⁺ and 8 mM NH₄⁺. A mole fraction mix of 2 mM K⁺:8 mM NH₄⁺ produced a greater inhibition of basolateral membrane K⁺ current than pure K⁺ or NH₄⁺ alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive ³⁶Cl^- uptake, e.g. NKCC-1. No anomalous effect was observed on Na⁺/K⁺-ATPase current. Both NKCC-1 and Na⁺/K⁺-ATPase activity were elevated in 10 mM NH₄⁺ with respect to 10 mM K⁺. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral NH₄⁺ on cAMP-stimulated transport is dependent on the [K⁺]₀/[NH₄⁺]_o ratio at the basolateral membrane.