Amino acid deprivation activates the amino acid response (AAR) pathway that enhances transcription of genes containing an amino acid response element (AARE). The present data reveal a quantitative difference in the response to deprivation of individual amino acids. The AAR leads to increased eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and ATF4 translation. When HepG2 cells were deprived of an individual essential amino acid, p-eIF2alpha and activating transcription factor 4 were increased, but the correlation was relatively weak. Complete amino acid starvation in either Earle's balanced salt solution or Krebs-Ringer bicarbonate buffer (KRB) resulted in activation of transcription driven by a SNAT2 genomic fragment that contained an AARE. However, for the KRB, a proportion of the transcription was AARE-independent suggesting that amino acid-independent mechanisms were responsible. Therefore, activation of AARE-driven transcription is triggered by a deficiency in any one of the essential amino acids, but the response is not uniform. Furthermore, caution must be exercised when using a medium completely devoid of amino acids.