Abstract 1. 1. A previous report demonstrated an increase in aspartate transcarbamoylase activity (carbamoylphosphate: l -aspartate carbamoyltransferase, EC 2.1.3.2) upon incubation of an homogenate of Escherichia coli with ATP, GTP, phosphoenolpyruvate, amino acids and magnesium acetate. The object of this study was to ascertain whether enzyme synthesis de novo or enzyme activation was responsible for the increase in aspartate transcarbamoylase activity. 2. 2. Utilizing density labeling and subsequent equilibrium density centrifugation it was demonstrated that the increase in aspartate transcarbamoylase activity was not due to synthesis de novo of this enzyme or to extensive completion of preexisting peptides. 3. 3. It was shown that incubation of an Escherichia coli homogenate with ATP, GTP, phosphoenolpyruvate, amino acids and Mg2+ resulted in a decrease in the apparent K m of aspartate transcarbamoylase for aspartate while the υ max changed little. 4. 4. Crystalline aspartate transcarbamoylase behaved differently than the enzyme in crude homogenates. Incubation of the crystalline enzyme with ATP, GTP and Mg2+ caused a threefold increase in υ max . This is the first report of an allosteric effector altering the υ max of aspartate transcarbamoylase. 5. 5. ATP, GTP and Mg2+ act in a synergistic manner and must be present together for maximum activation of aspartate transcarbamoylase. Previously published studies showed that ATP is an activator and GTP is an inhibitor of this enzyme. 6. 6. Mg2+ modify the effect of purine nucleotides on aspartate transcarbamoylase activity. GTP in equimolar concentration with Mg2+ had little effect on enzyme activity, while ATP and equimolar Mg2+ together were much better activators than ATP alone. 7. 7. The activation of aspartate transcarbamoylase by ATP and GTP in the presence of Mg2+ reported here may be of biological significance in regulating the relative pool size of pyrimidine nucleotides and purine nucleotides of Escherichia coli.