Amino acid deprivation of mammalian cells causes a significant enhancement in gene expression for a number of important cellular activities, among these is included asparagine synthetase (AS). A full length cDNA clone for rat AS was isolated previously from a subtracted cDNA library enriched for amino acid-regulated sequences. The present report summarizes the use of the AS cDNA to investigate the amino acid-dependent regulation of AS mRNA in normal rat liver and Fao hepatoma cells. In response to complete amino acid starvation, there was an increase in steady state AS mRNA content. Three species of mRNA, approximately 2.0, 2.5 and 4.0 kb, were detected and each was simultaneously regulated to the same degree. In hepatoma cells the increased AS mRNA content was prevented by either actinomycin D or cycloheximide. Partial repression of the AS mRNA content was maintained by the presence of a single amino acid in the culture medium, but the effectiveness varied. Glutamine effectively repressed the AS mRNA content, even at a concentration 10 times below its plasma level. Conversely, depletion of selected single amino acids from complete culture medium also caused up-regulation. A role for tRNA charging in the signalling mechanism was suggested by the observation that the addition of histidinol, an inhibitor of histidinyl tRNA synthetase, caused an increase in AS mRNA content when added to complete medium. The increased AS mRNA is associated with polysomes and is actively translated. The data indicate that nutrient regulation of the rat AS gene occurs by a general control mechanism that is responsive to the availability of selected individual amino acids.