Insulin regulation of K + (Rb + ) transport was investigated in cultured human fibroblasts using a non-radioactive method which allows the simultaneous determination of the intracellular concentration of other monovalent cations. Insulin stimulated Rb + influx through the Na + ,K + -ATPase and the Na + /K + /Cl - contransporter in human fibroblasts. Insulin stimulation was very rapid and maximal effect was observed within 10 min. Insulin stimulation of Rb + uptake via the Na + ,K + -ATPase and the Na + /K + /Cl - cotransporter was dose-dependent, with half-maximal stimulation at 2-3 nM of hormone. Insulin increased the V m a x of both transporters involved, affecting only minimally their K m . In other cells, insulin stimulates the Na + ,K + -pump by increasing Na + availability through the Na + /H + exchanger. In human fibroblasts, insulin stimulation of Na + ,K + -ATPase occurred in the presence of ethyl-isopropyl amiloride, an inhibitor of the Na + /H + exchanger, and without sustained changes in intracellular [Na + ]. By contrast, insulin action on Na + ,K + -ATPase was impaired by the protein kinase inhibitors staurosporine and genistein. These results indicate that, in human fibroblasts, insulin stimulates both the Na + ,K + -ATPase and the Na + /K + /Cl - cotransporter, that stimulation of the Na + ,K + -ATPase occurs in the absence of changes in intracellular [Na + ], and that protein kinase activity is essential for this insulin action.