l-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine transporter ASCT2, although it is known that it also inhibits other sodium-dependent amino acid transporters. In a panel of human cancer cell lines, which express the system l transporters LAT1 and LAT2, GPNA inhibits the sodium-independent influx of leucine and glutamine. The kinetics of the effect suggests that GPNA is a low affinity, competitive inhibitor of system l transporters. In Hs683 human oligodendroglioma cells, the incubation in the presence of GPNA, but not ASCT2 silencing, lowers the cell content of leucine. Under the same conditions the activity of mTORC1 is inhibited. Decreased cell content of branched chain amino acids and mTORC1 inhibition are observed in most of the other cell lines upon incubation with GPNA. It is concluded that GPNA hinders the uptake of essential amino acids through system l transporters and lowers their cell content.