These experiments were designed to determine the relative contributions of active NH4+ transport and voltage-driven NH4+ diffusion to direct NH4+ absorption by the medullary thick ascending limb of the rat. Medullary thick ascending limbs were perfused in vitro with solutions containing 25 mM HCO3 and 4 mM total ammonia. Under steady-state conditions, the lumen-positive transepithelial voltage (VT) was not sufficient to account for the observed decrease in lumen NH4+ concentration, consistent with active absorption of NH4+. Flux calculations based on VT and measured NH4+ permeability (6 x 10(-5) cm/s) indicate that the majority (at least 65%) of total ammonia absorption is due to active transport of NH4+. The remainder of NH4+ absorption can be accounted for by voltage-driven diffusion. Increasing the potassium concentration from 4 to 24 mM in perfusate and bath markedly inhibited total ammonia absorption but did not affect VT, NH4+ permeability, or HCO3 absorption. These results are consistent with inhibition of the active component of NH4+ absorption by potassium. The active NH4+ absorption is likely mediated by cotransport of Na+, NH4+, and Cl- across the apical cell membrane. Inhibition of active NH4+ absorption by an increase in potassium concentration may be due, in part, to competition between NH4+ and K+ for a common binding site on the Na+ -K+ -2Cl- cotransport system.