Intracellular chloride activities, (Cl)c, and the electrical potential difference across the mucosal membrane, ψmc, 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 ψmc averages -69mV 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 ψmc 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 (32mV) by this tissue, and (2) this accumulation is coupled to and energized by the entry of Na down its steep electrochemical potential difference. © 1979 Springer-Verlag New York Inc.