This chapter focuses on a group of loop diuretics that are chemically similar to furosemide. More specifically, issues regarding the chemistry, structure-activity relationships, and methods for evaluating the activity of loop diuretics are discussed. The loop diuretics are a group of substituted sulfamyl benzoic acid derivatives that include furosemide, bumetanide, and related compounds. Structure-activity studies using 3-aminobenzoic acid derivatives have focused on substitutions at three positions on the molecule. Diuretic-type drugs can be divided into 3 major categories; (1) carbonic anhydrase inhibitors, (2) thiazide-type diuretics, and (3) loop or high-ceiling diuretics. This chapter also addresses the mechanism of action of these compounds in blocking C1- transport and their use in the molecular characterization of the Na+/K+/2C1– co-transport system which is specifically inhibited by this group of diuretics. The efficacy and order of potency of loop diuretic compounds serve as important criteria in distinguishing the Na+/K+/2CI- cotransport system from other coexisting transport mechanisms present in cell membranes.