1. Binding between the radiolabeled loop-acting diuretics ([14C]frusemide, [14C]ethacrynic acid and [3H]bumetanide) and human Tamm–Horsfall glycoprotein or human serum albumin in vitro was evaluated by equilibrium dialysis.
2. The diuretic action and binding to urinary Tamm–Horsfall glycoprotein of the radiolabeled diuretics in vivo, after intravenous administration, were examined in rabbits.
3. In vitro, all three radiolabeled diuretics bound strongly to human serum albumin, but not to Tamm–Horsfall glycoprotein.
4. Radiolabeled frusemide and bumetanide, but not ethacrynic acid, caused a diuresis in rabbits, but no binding between the drugs and Tamm–Horsfall glycoprotein was seen in vivo.
5. Binding to Tamm–Horsfall glycoprotein does not appear to be an important mechanism in the action of loop diuretics.