Insulin stimulates K+ transport by the Na+-K+-ATPase in human fibroblasts. In other cell systems, this action represents an automatic response to increased intracellular [Na+] or results from translocation of transporters from an intracellular site to the plasma membrane. Here we evaluate whether these mechanisms are operative in human fibroblasts. Human fibroblasts expressed the alpha(1) but not the alpha(2) and alpha(3) isoforms of Na+-K+-ATPase . Insulin increased the influx of Rb+, used to trace K+ entry, but did not modify the total intracellular content of K+, Rb+, and Na+ over a 3-h incubation period. Ouabain increased intracellular Na+ more rapidly in cells incubated with insulin, but this increase followed insulin stimulation of Rb+ transport. Bumetanide did not prevent the increased Na+ influx or stimulation of Na+-K+-ATPase. Stimulation of the Na+-K+-ATPase by insulin did not produce any measurable change in membrane potential. Insulin did not affect the affinity of the pump toward internal Na+ or the number of membrane-bound Na+-K+-ATPases, as assessed by ouabain binding. By contrast, insulin slightly increased the affinity of Na+-K+-ATPase toward ouabain. Phorbol esters did not mimic insulin action on Na+-K+-ATPase and inhibited, rather than stimulated, Rb+ transport. These results indicate that insulin increases the turnover rate of Na+-K+-ATPases of human fibroblasts without affecting their number on the plasma membrane or modifying their dependence on intracellular [Na+].