Chronic K loading and Na deficiency increase the capacity of colonic cells to secrete K and are characterized by increases in Na-K-ATPase activity and, as recently shown by our laboratory, the area of basolateral membrane. The present experiments were performed in rat colon to evaluate the quantitative and qualitative changes that occur in the enzymes after induction of K adaptation. Changes in specific binding of [3H]ouabain to a plasma membrane-rich fraction of colonic mucosal cells correlated directly with changes in enzyme activity. In Na-deficient animals the number of ouabain-binding sites increased from 63.6 in controls to 111.2 pmol/mg protein, while the KD for ouabain was unchanged. The Km for ATP was not altered in experimental animals, although Vmax rose twofold. Further studies showed no change in the activity of adenylate cyclase, a marker enzyme for basolateral membrane, in the plasma membrane-rich fraction from experimental cells, although Na-K-ATPase activity increased significantly. These data indicate that increased Na-K-ATPase activity in K-adapted colonic cells reflects an absolute increase in number of pump sites, due, at least in part, to an increase in area of basolateral cell membrane. In addition, in sodium-deprived animals the present data suggest an increase in the density of K pumps in basolateral cell membrane.