Intracellular chloride activities, (Cl)c, and the electrical potential difference across the mucosal membrane, psimc, were determined in the isolated small intestine of the winter flounder, using Cl-selective and conventional (KCl-filled) microelectrodes. In the presence of a Na-containing buffer psimc averages -69 mV and (Cl)c averages 24 mM, a value that is 3.4 times that predicted for an equilibrium distribution across the mucosal membrane. On the other hand, when the tissue is then perfused with Na-free buffer, (Cl)c slowly falls to a value that does not differ significantly from that predicted for an equilibrium distribution, and psimc depolarizes significantly. Finally, when the tissue is again bathed in the Na-containing buffer, (Cl)c rapidly returns to a value well above equilibrium. These results, together with those of Frizzell et al. (J. Membrane Biol. 46:27, 1979), provide direct evidence that: (1) Cl is accumulated against its electrochemical potential difference (32 mV) by this tissue, and (2) this accumulation is coupled to and energized by the entry of Na down its steep electrochemical potential difference.