The effects of amino acid imbalance, created by decreasing the concentrations of specific amino acids in culture media, on intracellular protein synthesis were compared with the findings obtained by restricting all amino acids. Protein-synthesis levels were estimated by measuring the uptake rates of 14C-labeled amino acids by cultured rat hepatoma AH109A cells. The inhibitory activity of a 0.1-fold dilution of valine in DM-160 medium was higher than that resulting from the dilution of all amino acids by 0.1-fold. On the other hand, the dilution of other essential amino acids was not as effective as the dilution of all amino acids, whereas the concurrent dilution of valine and leucine was found to be equivalent in effectiveness to the latter. Protein-synthesis levels in 0.3, 0.1, 0.03, and 0.01 mM valine were maximum at 0.9, 0.3, 0.09, and 0.03 mM leucine, respectively. Valine transport into the cell was found to be inversely proportional to the extracellular leucine concentration. DL-Norvaline and DL-norleucine were not effective. Valine-leucine interactions were suggested to be involved in intracellular protein synthesis.