Muscle-stripped piglet colon was used to evaluate changes in short-circuit current (I_sc) as an indicator of anion secretion. Mucosal exposure to Escherichia coli heat-stable (STa) or heat-labile enterotoxins (LT) stimulated I_sc by 32 ± 5 and 42 ± 7 µA/cm², respectively. Enterotoxin-stimulated I_sc was not significantly affected by either 4,4'-diaminostilbene-2,2'-disulfonic acid or CdCl₂, inhibitors of Ca²⁺-activated Cl channels and ClC-2 channels, respectively. Alternatively, N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), a compound that inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl secretion, reduced I_sc by 29 ± 7 and 34 ± 11 µA/cm², respectively. Two additional diarylsulfonylurea (DASU)-based compounds were evaluated for their effects on enterotoxin-stimulated secretion. The rank order of potency for inhibition by these three closely related DASU structures was identical to that observed for human CFTR. The degree of inhibition by each of these compounds was similar for both STa and LT. The structure- and concentration-dependent inhibition shown indicates that CFTR mediates both STa- and LT-stimulated colonic secretion. Similar structure-dependent inhibitory effects were observed in forskolin-stimulated rat colonic epithelium. Thus DASUs compose a family of inhibitors that may be of therapeutic value for the symptomatic treatment of diarrhea resulting from a broad spectrum of causative agents across species.