The mechanism for 201Tl+ uptake was studied in cultured human glioma cell lines. Ouabain (1 mM) decreased the uptake at steady-state to 60%, but the rate of uptake was faster in the presence of ouabain. Addition of non-radioactive Tl+ (to a K+-free medium) decreased the uptake, but much less than expected for a system limited by the number of transport sites. Changes in K+ concentration of incubation medium affected the 201Tl+ uptake as predicted by the electrochemical equilibrium (Nernst equation). Using the uptake in isotonic KCl as a reference for membrane potential (0 mV), the calculated membrane potential was -75 mV in a medium with 3.0 mM K+. The Tl+-flux constants and the membrane permeabilities for Tl+ and K+ were calculated from the rate of uptake and from wash-out experiments. This is a new method for membrane potential and permeability studies in cell populations. The mechanism for 201Tl+ uptake is relevant for the clinical interpretation of 201Tl+ scintigraphy.