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 ₁ but not the ₂ and 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⁺].