In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na⁺,K⁺,Cl⁻-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH ₄ ⁺ on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH ₄ ⁺ inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K⁺. Inhibition of bumetanide-sensitive rubidium uptake by NH ₄ ⁺ was competitive and an apparentK _i of 1.9 mmol/liter was found Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH ₄ ⁺ . A 100-mmol/liter NH₄Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH ₄ ⁺ is accepted by the K⁺ site of the Na⁺,K⁺,Cl-cotransport system and that the transporter can function in a Na⁺, NH ₄ ⁺ ,2Cl mode. Since the affinity of the transporter for NH ₄ ⁺ lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na⁺, NH ₄ ⁺ 2Cl-cotransport can contribute to the NH ₄ ⁺ reabsorption in this tubular segment.