The binding of the new diuretic piretanide and two other diuretics (furosemide and bumetanide) to serum proteins (especially albumin) was studied using the in vitro equilibrium dialysis method. Piretanide and bumetanide, similar to each other in chemical structure, were almost equal in the binding constant to purified albumin, while the binding constant of furosemide was greater than that of the other two diuretics. The binding of these loop diuretics in the dilutions of the sera from healthy adults and patients with renal failure or liver disease was also studied by the same method. Protein binding of the drugs was markedly reduced in patients with renal failure. The cause could only partly be explained by some structural changes in the serum albumin molecule of these patients and influences of endogeneous substances present in serum.