We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2CI--cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LKl) exhibited alterations in the Na+,K+,2CI--cotransport system. LK, cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, humetanide-sensitive potassium influx, measured by 86Rbi uptake, was increased in the LK1 cells (increase in vmaX). K , values of the cotransporter in HeLa cells and LKI mutants revealed similar properties for 86KbL and 12Naf uptake. In addition, (3H)-burnetanide binding studies were carried out on intact HeLa cells; 1.7 pmolimg protein (3H)-bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding siteskell. LK, cells present, 1.44 pmolimg protein, specifically bound (3H)-bumetanide and, respectively, 137,000 binding sitesicell. The LK1 cells also exhibited an increase in the number of (3H)-ouabain binding sites as well as an increase in the activity of the Na+,K+-ATPase, expressed as a function of ouabain-sensitive s6Rb+ uptake. Furthermore, LK, cells were different in the concentrations of intracellular Na' ( t ) and K + ( ) when compared to the HeLa parental cells. When grown in low K + medium (0.2 mM K+), protein content and cell volume were increased in the LK, cells, while the DNA content was not significantly different between both cell lines.