Inhibition of gastric acid secretion by the cardiac glycoside, ouabain, is considerably reduced by elevating external K+ as first shown by Davenport (Proc. Soc. Exp. Biol. Med. 110: 613-615, 1962). To determine the possible role of K+ in this effect, we measured [3H]ouabain binding in isolated bullfrog gastric mucosa. Uptake of the labeled drug showed two components: one that saturated at 0.36 pmol ouabain per milligram wet weight and one that was linear with the external ouabain concentration. The former component is considered to represent specific binding to Na+-K+-ATPase; activity of this enzyme system in mucosal homogenates was 0.2 mumol per milligram protein per hour. Increase of K+ in the nutrient bathing solution from 3 to 30 mM, or replacement of Na+ by K+ in the secretory bathing solution, largely reversed inhibition of acid secretion by ouabain but did not affect maximum specific binding. The results fit a model in which Na+-K+-ATPase is normally required in oxyntic cells to maintain a high K+ level, which in turn supports exchange of K+ for H+ at the apical surface.