Alanine is a powerful stimulator of hepatic protein synthesis whose mechanism of action has not yet been ascertained. The present work aimed to elucidate whether rate changes in ion fluxes accompanying the transport of this amino acid could play a role in the stimulation of protein synthesis. In perfused livers, the utilization of alanine produced a net uptake of (\mathrm{K}^{+})of (1.5 \mu \mathrm{mol} / \mathrm{min} /) liver, a progressively increasing efflux of (\mathrm{Ca}^{2+}) to reach a maximum of (0.9 \mu \mathrm{mol} / \mathrm{min} /) liver, and alkalization of the extracellular medium. Inhibition of (\mathrm{Na}^{+} / \mathrm{K}^{+})exchange by ouabain reversed only the uptake of (\mathrm{K}^{+}), indicating that this is the main way for the efflux of (\mathrm{Na}^{+})cotransported with alanine. In isolated hepatocytes, the uptake of alanine increased the intracellular content of (\mathrm{K}^{+})and the cell volume. The following observations suggest that these changes, and not an increased intracellular concentration of (\mathrm{Na}^{+}), are associated with the stimulation of protein synthesis: 1) Ouabain inhibited the alanine stimulation of (\mathrm{L}-\left[{ }^{3} \mathrm{H}\right])-valine incorporation into protein without altering the basal rate of protein labeling; 2) ouabain had no effects on alanine uptake indicating that (\mathrm{Na}^{+})influx is not involved in the alanine stimulation of protein synthesis; 3) disruption of (\mathrm{Na}^{+})gradient across the plasma membrane by specific ionophores, monensin and gramicidin D, inhibited both basal and alanine-stimulated protein synthesis, but substitution of extracellular (\mathrm{Na}^{+})by (\mathrm{K}^{+})did not prevent the stimulatory action of alanine. The observation that hypotonic buffer enhanced protein synthesis to the same degree than alanine in liver cells indicates that alanine-induced cell swelling could be sufficient to stimulate protein synthesis. ① 1995 Wiley-Liss, Inc.