Equilibrium and kinetic studies of the interaction of the modifier analog 5-bromocytidine triphosphate (BrCTP) with native aspartate transcarbamylase and its regulatory subunit have been carried out utilizing difference spectroscopy and the temperature-jump method. Binding constants were determined under a variety of conditions. A single relaxation process was observed with relaxation times in the range of 0.1-1 msec for solutions containing BrCTP and native ATCase, both in the presence and absence of carbamyl phosphate and the aspartate analog, succinate, and for solutions containing BrCTP and the regulatory subunit. Rapid mixing experiments revealed that the entire binding process was complete in less than 5 msec. The concentration dependence of the relaxation times indicates a bimolecular reaction is rate determining in the case of the regulatory subunit, and probably for the native enzyme in the absence of substrates. In the presence of carbamyl phosphate and succinate, a conformational change of the native enzyme becomes rate limiting; a corresponding change in mechanism is not observed with the regulatory subunit. These data are consistent with two types of allosteric control mechanisms: a mechanism similar to that of Monod et al. involving two conformational states of the enzyme and one involving a rapid bimolecular reaction followed by a relatively slow conformational change. The concentration dependence of the relaxation time in the presence of phosphate is best explained by the two state mechanism. Therefore, this type of mechanism is most consistent with all of the data.