The neutral amino acid transport activity, System A, is enhanced by amino acid limitation of mammalian cells. Of the three gene products that encode System A activity, the one that exhibits this regulation is SNAT2 (sodium-coupled neutral amino acid transporter 2). Fibroblasts that are deficient in the amino acid response pathway exhibited little or no induction of SNAT2 mRNA. Synthesis of SNAT2 mRNA increased within 1-2 h after amino acid removal from HepG2 human hepatoma cells. The amino acid responsive SNAT2 genomic element that mediates the regulation has been localized to the first intron. Increased binding of selected members of the ATF (activating transcription factor) and C/EBP (CCAAT/enhancer-binding protein) families to the intronic enhancer was established both in vitro and in vivo. In contrast, there was no significant association of these factors with the SNAT2 promoter. Expression of exogenous individual ATF and C/EBP proteins documented that specific family members are associated with either activation or repression of SNAT2 transcription. Chromatin immunoprecipitation analysis established in vivo that amino acid deprivation led to increased RNA polymerase II recruitment to the SNAT2 promoter.