Transcription initiation sites of the asparagine synthetase gene were investigated in human hepatoma cells after amino acid limitation by incubation in amino acid-complete minimal essential medium or medium lacking histidine. Cells incubated in complete minimal essential medium had mRNA transcripts with starting positions spanning across the 69 nucleotides immediately upstream of a previously designated transcription start site (+1), whereas the majority of mRNA transcripts started at nucleotide (+1) in cells incubated in histidine-free medium. Similar results were obtained regardless of whether the analysis was by 5' rapid amplification of cDNA ends or a ribonuclease protection assay. Low ASNS mRNA expression in amino acid-complete medium was associated with the wide range of initiation sites, whereas preferred alignment of the general transcription machinery at nucleotide (+1), observed in the amino acid deprived condition, was associated with a concurrent increase in transcription activity. To our knowledge, these results are the first example in a mammalian cell of transcription start selection by nutrient availability.