When rat liver was pulse-labeled with [3H]valine in vivo, the nascent peptide on membrane-bound polysomes was found to be more highly labeled than that on free polysomes. Nascent peptides were purified from both classes of polysomes and, after hydrolysis, the amino acids were reacted with 14C-labeled 1-fluoro-2,4-dinitrobenzene. The specific activity of [3H]valine was determined from the [14C]-dinitrophenyl-[3H]valine after purification by two-dimensional thin layer chromatography. With this approach we found that the specific activity of [3H]valine in the nascent peptide of membrane-bound polysomes was more than twice that of free polysomes. Moreover, when rats were pretreated with a lysosomal protease inhibitor, the differences between the specific activities of valine in nascent peptides of the two classes of polysomes tended to decrease. Our results indicate the existence of two distinct pools for valine used for protein synthesis in liver cells; one serves as a precursor for the synthesis of secretory proteins on membrane-bound polysomes and the other as a precursor for the synthesis of intracellular proteins on free polysomes.