To characterize and localize a K+/H+ antiport mechanism in the renal medullary thick ascending limb (MTAL), membrane vesicles were isolated from a rat MTAL homogenate. K+/H+ antiport (in > out H+ gradient-stimulated 86Rb+ uptake) was abolished by barium and verapamil (apparent Ki of 55 microM) but unaffected by other K+ channel blockers such as quinidine and high amiloride concentrations. SCH 28080, a H+/K+-ATPase blocker, did not affect K+/H+ antiport. K+/H+ antiport activity was correlated positively with the enrichment factor of the membranes in the apical marker enzyme alkaline phosphatase (r = 0.875, p < 0.01) and negatively correlated with the enrichment factor in basolateral Na+/K+-ATPase (r = -0.665, p < 0.05). Moreover, a functional interaction occurred with Na+/H+ exchange (NHE) consistent with colocation of K+/H+ antiport and apical NHE-3, not basolateral NHE-1. K+/H+ antiport was shown by intracellular pH measurements to be inhibited by arginine vasopressin and 8-bromo-cAMP through cAMP-dependent protein kinase (protein kinase A) activation. These results demonstrate the presence of a K+/H+ antiport mechanism, which is inhibited by arginine vasopressin via protein kinase A, in the apical membrane of the MTAL.