In hepatocyte cultures, insulin stimulates cellular accumulation of K+, partly (approximately 20%) by net replacement of cell Na+, but largely (approximately 80%) by increasing the cell K++Na+ content, with a consequent increase in cell volume. An increase in cation content occurred within 5 min of exposure to insulin and was not secondary to metabolic changes. Insulin also increased the cation content, by increasing the Na+ content, in a K(+)-free medium or when K+ uptake was inhibited with 1 mM-ouabain. However, insulin did not increase the cation content in a Na(+)-free medium. The stimulation of glycogen synthesis by insulin, like the increase in cation content, was blocked in a Na(+)-free medium, but not when K+ uptake was inhibited. Hypo-osmotic swelling restored the stimulation of glycogen synthesis in a Na(+)-free medium, indicating that the lack of effect of insulin in the iso-osmotic Na(+)-free medium was not due to a direct requirement for Na+ for glycogen synthesis, but to a secondary mechanism, dependent on Na+ entry, that can be mimicked by hypo-osmotic swelling. Quinine increased cell volume further and caused a further increase in glycogen synthesis. The hypothesis that cellular uptake of K+ may be part of the mechanism by which insulin controls metabolism was discounted, because inhibition of K+ uptake does not block the metabolic effects of insulin [Czech (1977) Annu. Rev. Biochem. 46, 359-384]. The present results support the hypothesis that an increase in cell cation content, and thereby cell volume, rather than K+ uptake, is part of the mechanism by which insulin stimulates glycogen synthesis in hepatocytes.