Long-term supplementation of branched-chain amino acids (BCAA) improves hypoalbuminemia in patients with cirrhosis. Our previous findings have suggested that the binding of polypyrimidine-tract-binding protein (PTB) to rat albumin mRNA attenuates its translation. The aim of the present study was to investigate the role of PTB in the regulation of albumin synthesis by BCAA in human hepatoma cells. HepG2 cells were cultured in a medium containing no amino acids (AA-free medium), a medium containing only 1 amino acid (a BCAA: valine, leucine or isoleucine) or a medium containing all 20 amino acids (AA-complete medium). HepG2 cells cultured in AA-complete medium secreted much more albumin than cells cultured in AA-free medium, with no difference in albumin mRNA levels. In cells cultured in AA-free medium, nuclear export of PTB was observed, and the level of the albumin mRNA-PTB complex was greater than in cells cultured in AA-complete medium. Addition of amino acids stimulated nuclear import of PTB. However, addition of amino acids with rapamycin inhibited the nuclear import of PTB. The addition of leucine, but not of valine or isoleucine, to AA-free medium increased albumin secretion and stimulated the nuclear import of PTB. These data indicate that the mammalian target of rapamycin is involved in the regulation of PTB localization and that leucine promotes albumin synthesis by inhibiting the formation of the albumin mRNA-PTB complex.