The intracellular Cl- activity was determined in rat kidney proximal tubular cells in vivo, using single-barreled Cl- sensitive microelectrodes filled with Corning no. 477913 liquid ion exchanger resin to measure VCl and using - in separate experiments - conventional KCl-filled microelectrodes to measure the membrane potential, Vm. After correction for interference from other anions on VCl the intracellular Cl- activity averaged 13.1 mmol X l-1 SD +/- 4.5 mmol X l-1 (n = 96). This value is approximately two-fold higher than the intracellular equilibrium activity which can be calculated from the extracellular Cl- activity of 90-103 mmol X l-1 and Vm of -71.2 mV, SD +/- 4.9 mV (n = 23) to amount to 6.3 to 6.7 mmol X l-1. Since both cell membranes are permeable for Cl- ions, as concluded from luminal and/or peritubular Cl- substitution experiments, we conclude that the cellular Cl- accumulation above equilibrium results from transcellular active Cl- transport, the detailed mechanism of which is presently not known. From the slow decline of intracellular Cl- concentration after substitution of luminal Cl- by gluconate, however, we deduce that transcellular Cl- absorption is of minor importance in surface tubules of rat kidney under free flow and that the major part of transtubular Cl- flux is passive and paracellular.