The effect of insulin on rat adipocytes was studied in isotonic buffers (pH 7.4) containing NaCl, CaCl2, MgSO4, KCl, and bovine serum albumin but no phosphate or bicarbonate anions. In phosphate- and bicarbonate-free buffers the dose-response curve to insulin is shifted to the right, the effects of the hormone on hexose uptake, glucose metabolism, and inhibition of lipolysis being observed at much higher (nearly 2 orders of magnitude) concentrations of insulin. The insulin binding capacity of the cells is only slightly changed. The dose-response curve for isoproterenol which stimulates lipolysis in the same cell type is almost the same in both Krebs-Ringer bicarbonate buffer and phosphate- and bicarbonate-free buffers. The dose-response curves for agents that mimic the action of insulin such as wheat germ agglutinin or vanadate ions are also shifted to the right. The dose-response curve to insulin can be returned to "normal" by readdition of either bicarbonate or phosphate. Almost complete recovery is obtained at either 10 mM bicarbonate or 24 mM phosphate, respectively. External Ca2+ ions which are not required for the proper action of insulin in fat cells maintained in Krebs-Ringer bicarbonate buffer, become essential for insulin action in bicarbonate-free buffer. The study indicates that depletion of bicarbonate and, to a lesser extent, phosphate anions, interferes with an essential insulin-dependent post-binding event. Also, in bicarbonate-free medium, external Ca2+ ions are essential for insulin-mediated processes. The implications of this study to the mode of action of insulin, and to physiological and clinical states of insulin desensitization are discussed.