Kinetic studies of the interaction of native aspartate transcarbamylase with two aspartate analogs, succinate and L-malate, were carried out using the temperature-jump method. In the presence of saturating carbamyl phosphate, a single relaxation process was observed for the binding of succinate to the enzyme-carbamyl phosphate complex. The reciprocal relaxation time decreases with increasing succinate concentration, and can be quantitatively analyzed in terms of a concerted mechanism analogous to the model of Monod et al. (Monod J., Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88). Similar studies with L-malate revealed a relaxation process that also could be quantitatively analyzed in terms of the concerted mechanism. When both BrCTP (an allosteric inhibitor) and succinate were present with saturating carbamyl phosphate, two relaxation processes were observed, which were associated with two distinct conformational transitions of the enzyme induced by BrCTP and succinate, respectively. This result suggests that more than two conformational states of the enzyme are of importance in the allosteric regulation. A mechanism involving four conformational states is postulated to account for both the homotropic and heterotropic effects observed experimentally.