1. 1. The effects of monovalent cations on glucose uptake by isolated rat soleus muscle could be shown, simply by varying the concentrations of these cations in the incubation medium. Glucose uptake was stimulated by Na+ and inhibited by K+. Stimulation of glucose uptake by insulin did not depend on the presence of monovalent cations in the incubation medium. 2. 2. In order to demonstrate the effect of divalent cations, it was necessary to "strip" the endogenous Ca2+ and Mg2+ from the muscle, by preincubation for 15 min in Krebs-Henseleit HCO3- medium containing 5 mM EDTA. Glucose uptake by these EDTA-stripped muscles, measured in Ca2+/Mg2+-free medium, was depressed, and could not be increased by the addition of insulin. 3. 3. The basal uptake rate could be restored to normal by the addition of either Ca2+ (2.5 mM) or Mg2+ (1.1 mM); at a concentration of 0.1 mM, Ca2+, but not Mg2+, was still effective. Conversely, Mg2+, but not Ca2+, was required for the stimulation of glucose uptake by insulin. 4. 4. It is proposed that Ca2+ is somehow required for the structural integrity of the cell membrane, whereas Mg2+ probably participates in the mechanism whereby insulin promotes the binding of glucose to the sugar transport carrier. © 1970.