Inhibition of Na⁺/H⁺ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 {3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydro-chloride} was the most potent and specific NHE3 inhibitor with an IC₅₀ value of 0.02 µmol/l for the human isoform, whereas its IC₅₀ value for hNHE1 and rbNHE2 was 3.6 and @80 µmol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC₅₀>100 µmol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC₅₀ for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 µmol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.