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Summary Ouabain-insensitive, furosemide-sensitive Rb+ influx (JRb) into HeLa cells was examined as functions of the extracellular Rb+, Na+ and Cl- concentrations. Rate equations and kinetic parameters, including the apparent maximumJRb, the apparent values ofKm for the three ions and the apparentKi for K+, were derived. Results suggested that one unit molecule of this transport system has one Na+, one K+ and two Cl- sites with different affinities, one of the Cl- sites related with binding of Na+, and the other with binding of K+(Rb+). A 1:1 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of22Na+ and Rb+, and a 1:2 stoichiometry between those of Rb+ and36Cl-. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl- inhibited ouabain-insensitive Rb+ influx, whereas sulfamate and probably also gluconate did not inhibitJRb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na+ and one Cl- bind concurrently to their sites and then one K+ (Rb+) and another Cl- bind concurrently. 2) After completion of ion bindings Na+, K+(Rb-) and Cl- in a ratio of 1:1:2 show synchronous transmembrane movements.