We have studied the transcription of the argininosuccinate synthetase gene in cultured RPMI 2650 cells under conditions where the enzyme is subject to metabolite regulation and in canavanine-resistant variants (Canr1 cells) which overproduce the enzyme greater than 200-fold. When grown continuously in medium with citrulline substituted for arginine, the argininosuccinate synthetase activity of RPMI 2650 cells increases 5- to 10-fold. In these cells, expression of a transfected minigene containing the 5'-flanking region of the argininosuccinate synthetase gene was increased 20-fold by short term starvation for arginine and 10-fold by short term starvation for leucine. Levels of nuclear RNA from the first intron of the gene correlated with enzyme activity; i.e. RPMI 2650 cells cultured in arginine medium less than RPMI 2650 cells cultured in citrulline medium less than Canr1 cells. Run-off transcription experiments showed that the transcription of argininosuccinate synthetase increased in RPMI 2650 cells starved for either arginine or leucine. While expression of the minigene and the transcription rate for argininosuccinate synthetase were increased during 48 to 72 h of starvation, the endogenous enzyme activity did not increase in RPMI 2650 cells. Amino acid starvation did not affect the rate of transcription of argininosuccinate synthetase in Canr1 cells. The results indicate that the steady state levels of argininosuccinate synthetase expression in Canr1 cells and in citrulline-adapted RPMI 2650 cells are largely determined by the rate of transcription. The failure of increased transcription rate to correlate with increased enzyme activity during acute starvation for arginine or leucine may suggest the involvement of post-transcriptional regulatory mechanisms for argininosuccinate synthetase or may merely be due to amino acid deprivation. The finding that leucine starvation has effects similar to arginine starvation raises the question of whether mammalian cells have general control mechanisms which are similar to the general control of amino acid biosynthesis in Saccharomyces cerevisiae.