Abstract The results of the kinetic studies described in this report, of the binding studies in the accompanying paper, and of earlier investigations have led to the formulation of a kinetic mechanism for the carboxylation reaction catalyzed by phosphoenolpyruvate carboxykinase (PEP carboxykinase). Because of the complexity of this mechanism, no attempt was made to write an over-all rate equation for the initial velocity from which the experimental data might be interpreted. It proved more feasible to convert the pathway from free enzyme to the central complex (e-Mn2+-PEP-IDP-CO2) from a random one to an ordered, sequential one by designing experiments in which the concentrations of some species of the substrate were negligible relative to the concentrations of other species, allowing simplified rate equations to be written. It was possible, with this approach, to compile kinetic data which are consistent with the mechanism. Attempts to interpret the data on the basis of other mechanisms have led to inconsistencies. The most significant features of this mechanism are as follows. (a) The formation of a central complex consisting of enzyme, Mn2+, PEP, IDP, and CO2 proceeds by what may be described as a mixed ordered-random addition of the components. (b) PEP must bind to the enzyme before either IDP or CO2 in order to yield a kinetically active form of the enzyme. Although IDP and CO2 may bind without prior binding of PEP, they form inactive complexes that do not lead to the central complex, and they may be considered to be inhibitors with respect to PEP. (c) The binding of PEP proceeds in a random manner with respect to Mn2+ to form the ternary enzyme-Mn2+-PEP complex. Both free enzyme and enzyme-Mn2+ can bind PEP, although the affinity of the enzyme for PEP is greatly enhanced when Mn2+ is present, either on the enzyme or complexed with PEP. (d) The ternary enzyme-Mn2+-PEP complex may bind either IDP or CO2 to form a quaternary complex, which then binds the third substrate to form the central complex. (e) An alternative route to the quaternary enzyme-Mn2+-PEP-IDP complex involves the binding of MnIDP to the binary enzyme-PEP complex. (f) The central complex undergoes conversion to a new complex consisting of enzyme, Mn2+, oxalacetate, and ITP. The results of an experiment in which the reaction in the decarboxylation direction was carried out in H218O indicate that H2O does not participate directly in the reaction, since 18O was not incorporated into the products of the reaction (PEP and IDP). This result, in conjunction with the kinetic results reported herein and earlier findings, suggests that the chemical mechanism of the reaction is concerted.