For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPbeta (CCAAT/enhancer-binding protein beta) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPbeta genomic fragments containing the 8451 bp 5' upstream of the transcription start site did not contain amino-acid-responsive elements. However, deletion analysis of the genomic region located 3' downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acid-responsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPbeta enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPbeta promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPbeta itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPbeta, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.