SUMMARY1. Measurements were made of the binding of 73H]ouabain to a variety of cell types.2. Two components of binding could usually be distinguished: a component that saturated at low glycoside concentrations and a component that increased up to the highest ouabain concentrations examined.3. Detailed studies with HeLa cells and kidney slices from guinea-pigs showed that the saturable component is probably associated with inhibition of the Na pump. The main evidence for this is (a) at low concentrations of ouabain there is a close correspondence between the concentration of ouabain giving half-maximum binding and the concentration giving halfmaximum inhibition of the Na pump; (b) at low glycoside concentrations, binding precedes inhibition of the Na pump; (c) the rate of binding is very sensitive to external K ions, being highest in the absence of K; (d) binding is reversible and the release of ouabain is associated with reactivation of the Na pump, (e) binding is reduced in the absence of Na ions and in the presence of metabolic inhibitors; (f) binding has a Q10 of about 4; and (g) in the presence of Na and ATP, lysed HeLa cells bind a similar amount of ouabain and the binding is sensitive to K ions.4. The linear component of binding does not seem to involve the Na pump and it may reflect uptake of ouabain into the cell interior. It has a Q10 of 2-5 and is unaffected by K concentrations which have a large effect on the saturable component.5. Bound ouabain could be removed from HeLa cells by low pH, trichloroacetic acid, urea, high temperatures and 100 % ethanol. These agents did not distinguish between the two components of binding. 6. Criteria are developed for estimating the number of Na pumping sites in cells and the data for ouabain-binding to a number of cells is compared * Present address: