Amino acids are key regulators of protein synthesis in liver. However, it remains to be determined whether amino acids stimulate synthesis of all or certain specific liver proteins. No techniques are currently available to simultaneously measure synthetic rates of several individual proteins. Here we report studies performed on rat hepatocyte primary cultures in which we used metabolic labeling with [¹⁴C] leucine, two dimensional gel electrophoresis (2DGE), and tandem mass spectrometry to identify proteins that showed increased leucine incorporation when high amino acid levels were present in the media. Rat hepatocytes were isolated by in situ collagenase perfusion, cultured in serum-free medium containing insulin, and incubated for 2, 4, and 8 hours in media of standard and high amino acid concentrations. SDS-PAGE and 2DGE were performed to separate proteins from cell lysates. Proteins that consistently showed increased synthesis on triplicate cultures, as detected by phosphor imaging of gels, were identified by tandom mass spectrometry. The combination of these approaches enabled the detection of 16 specific liver proteins whose synthetic rates were enhanced by increased amino acid concentration. These proteins are involved in specific functions such as translation intiation, protein folding and modification, oxidative phosphorylation, antioxidant defense, signal transduction, and transport, as well as cell motility and tissue integrity. No quantitative changes for any of these proteins were detected by gel staining, indicating that no detectable changes in protein concentration occurred. In contrast, measurable changes in synthetic rates occurred in 16 proteins. In conclusion, amino acids stimulate the synthesis of several liver proteins with important cellular functions.