It is not known whether factors other than Na transport are involved in generating the electrical potential difference (PD) across the human colonic wall. Therefore, experiments were performed in which the effect of amiloride on PD and ion transport was evaluated in the in vitro short-circuited human colon. In control periods the short-circuit current (Isc) was 2.9 +/- 0.3 (mean +/- S.E.M.) muEq per hr per cem2, while the corresponding net transfer of Na and Cl was 4.6 +/- 0.4 and 1.4 +/- 0.1 respectively. The residual flux was insignificantly different from zero. Amiloride caused a prompt, but reversible, decrease in Isc, PD, and conductance when added to the mucosal sie,dbut only a relatively small reduction of the mucosa to serosa fluxes of Na occurred. Bidirectional Cl fluxes were unchanged while the residual flux increased significantly. It is proposed, therefore, that some ions other than Cl (presumably mucosa to serosa fluxes of H and/or serosa to mucosa fluxes of HCO3) are the main counter ions for actively transported Na in the large bowel. Experiments performed in vivo showed that the rectal PD decreased exponentially from -46 mV +/- 0.8 TO -27 MV +/- 0.3 (mean +/- S.E.M.) following rectal instillation of 10-3 M amiloride. The half-time required for this effect was less than 13 seconds.