The group of "high ceiling" diuretics consists of a variety of chemically different potent diuretic and saluretic substances. Appart from a few exemptions direct evidence for an action of these substances in the thick ascending limb of the loop of Henle (TAL) is still lacking. For furosemide, we have reported recently that it inhibits most likely the Na+-2 Cl--K+ cotransport system present in the lumen membrane of the TAL. The present study tests: 1. whether other "high ceiling" diuretics have a similar site and mode of action, and 2. how modifications of the furosemide molecule alter the inhibitory potency. Isolated cortical TAL (cTAL) segments (n = 185) of rabbit kidneys were perfused in vitro. The equivalent short circuit current (Isc = transepithelial PD/transepithelial resistance), as a measure of active salt transport was correlated to the dose of 64 substances. Several diuretics, such as 2-aminomethyl-4-(1,1-dimethyl-ethyl)-6-iodophenol hydrochloride (MK 447), hydrochlorothiazide, muzolimine, etozoline, tizolimide, amiloride, and triamterene were ineffective both from the lumen and basolateral side at concentrations as high as 10(-4) - 10(-3) mol X 1(-1). The phenoxyacetic acids ethacrynic acid, indacrinone (MK 196), and to less an extend tienilic acid were inhibitory active. They differed from furosemide in one or more of the following criteria: delayed onset, incomplete reversibility, stronger action from the bath, different slope of the dose response curve. Similarly, 1-ozolinone acted stronger from the bath. In contrast, the diuretics of the furosemide type and related compounds (bumetanide and piretanide) showed rapid onset and complete reversibility of inhibition. These substances acted stronger from the lumen. The individual positions in the benzyl ring of the diuretics were differently affected by substitutions, leading to parallel shifts in the dose response curves with halfmaximal inhibition at concentrations ranging between 8 X 10(-8) to greater than 10(-4) mol X 1(-1). For these substances the calculated Hill coefficients were close to unity: 0.96 +/- 0.05. We conclude that the so called "high ceiling" or "loop" diuretics consist of at least 3 groups: 1. drugs that do not interfere with the active salt transport in the cTAL segment, 2. drugs that interfere by so far not characterised mechanisms, and 3. drugs of the furosemide type which inhibit the Na+-2 Cl--K+ cotransport system in the lumen membrane of the cTAL segment.