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Experiments were designed to characterize an active, electrogenic transport of NH(4)(+) ions across the colonic epithelium of the domestic fowl (Gallus gallus). Colonic segments were isolated and stripped of underlying muscle. The mucosal epithelia were mounted in Ussing chambers and voltage-clamped to measure the short-circuit currents (I(SC)) associated with transport. Bilateral addition of NH(4)(+) caused a dose-dependent outward current (negative I(SC)), with a Km of 34+/-8 mM and a maximal current response of 311+/-47 microA cm(-2) (12+/-2 microEq cm(-2) h(-1)). A similar effect was seen with unilateral addition of NH(4)(+) to the serosal (s) side, but not with mucosal (m) addition. Pre-treatment with 10(-4) M amiloride exposed a net outward (negative) I(SC), and serosal NH(4)(+) addition further increased this outward current with a Km of 53+/-24 mM. Decreasing the bath pH from 7.3 to 6.0 did not affect the I(SC) response to NH(4)(+). Unidirectional NH(4)(+) flux measurements revealed a net secretory flux (8.8+/-3.1 micromol cm(-2) h(-1) s-m, versus 2.6+/-1.4 micromol cm(-2) h(-1) m-s). Furthermore, the secretory flux closely matched the resulting change in I(SC) with serosal NH(4)(+), showing that the transepithelial flux of NH(4)(+) could account for the outward current response. Addition of 50 nM bafilomycin A to the mucosal solution completely eliminated serosal to mucosal NH(4)(+) transport, implicating an apical V-type H(+)-ATPase in this transport process. The I(SC) response to NH(4)(+) was partially inhibited by ouabain, a blocker of the Na(+)/K(+)-ATPase, but only minimally affected by bumetanide, an inhibitor of the serosal Na(+)-K(+)-2Cl(-) cotransporter. Active NH(4)(+) extrusion across the mucosal membrane, combined with low permeability to NH(3) in this tissue, allow for maintenance of steep ammonia gradients across the colonic epithelium and protection from ammonia toxicity. Furthermore, these studies indicate that the hen colon may be a useful