Amino acids are essential for activating the mammalian target of rapamycin (mTOR) kinase, which regulates protein translation, cell growth, and autophagy. The study reveals that the uptake of L-glutamine is the rate-limiting step in the activation of mTOR by essential amino acids (EAA) and growth factors. L-glutamine is transported into cells via the SLC1A5 transporter, and its subsequent efflux is regulated by the bidirectional transporter SLC7A5/SLC3A2, which exchanges intracellular L-glutamine for extracellular L-leucine. This bidirectional transport is critical for mTOR activation, as the efflux of L-glutamine allows the uptake of EAA, leading to the activation of S6K1 and subsequent mTOR signaling. The study shows that the availability of L-glutamine is essential for mTOR activation, and its absence leads to autophagy. Additionally, certain tumor cell lines with high basal levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. The findings highlight the role of L-glutamine and its transporters in regulating mTOR, translation, and autophagy to coordinate cell growth and proliferation. The study also demonstrates that the bidirectional transport of L-glutamine and EAA is essential for mTORC1 signaling and that the regulation of L-glutamine levels is crucial for maintaining cellular homeostasis. The results suggest that the interplay between L-glutamine and EAA transporters is a key mechanism by which cells regulate mTOR signaling and autophagy.Amino acids are essential for activating the mammalian target of rapamycin (mTOR) kinase, which regulates protein translation, cell growth, and autophagy. The study reveals that the uptake of L-glutamine is the rate-limiting step in the activation of mTOR by essential amino acids (EAA) and growth factors. L-glutamine is transported into cells via the SLC1A5 transporter, and its subsequent efflux is regulated by the bidirectional transporter SLC7A5/SLC3A2, which exchanges intracellular L-glutamine for extracellular L-leucine. This bidirectional transport is critical for mTOR activation, as the efflux of L-glutamine allows the uptake of EAA, leading to the activation of S6K1 and subsequent mTOR signaling. The study shows that the availability of L-glutamine is essential for mTOR activation, and its absence leads to autophagy. Additionally, certain tumor cell lines with high basal levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. The findings highlight the role of L-glutamine and its transporters in regulating mTOR, translation, and autophagy to coordinate cell growth and proliferation. The study also demonstrates that the bidirectional transport of L-glutamine and EAA is essential for mTORC1 signaling and that the regulation of L-glutamine levels is crucial for maintaining cellular homeostasis. The results suggest that the interplay between L-glutamine and EAA transporters is a key mechanism by which cells regulate mTOR signaling and autophagy.