Background: In this study, the membrane mechanisms of hyperkalemia caused by trimethoprim-sulfamethoxazole (TMP-SMX) combination antibiotics were assessed in the cortical collecting duct (CCD). Methods: We used the microelectrode technique and flux measurements, and examined the effects of TMP and SMX on electrical properties of the apical and basolateral membranes in the rabbit CCD perfused in vitro. Results: TMP in the lumen caused increases in apical membrane voltage, fractional apical membrane resistance (fR<sub>A</sub>), and transepithelial resistance (R<sub>T</sub>), all effects which were completely inhibited by luminal amiloride, but not by luminal Ba<sup>2+</sup>. The luminal TMP inhibited both net Na<sup>+</sup> reabsorption and K<sup>+</sup> secretion in the CCD. TMP in the bath slightly but significantly depolarized transepithelial voltage and basolateral membrane voltage without influencing fR<sub>A</sub> or R<sub>T</sub>. SMX in the lumen or bath had no effect on barrier voltages or resistances. Conclusion: TMP mainly acts on the apical membrane of the CCD, inhibits the amiloride-sensitive macroscopic Na<sup>+</sup> conductance in this membrane, and thereby decreases the net driving force for K<sup>+</sup> exit across the membrane, resulting in an inhibition of K<sup>+</sup> secretion. SMX in the lumen or bath had no effect on the CCD.