In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.