Phosphoenolpyruvate (PEP) carboxylase from Escherichia coli is an allosteric enzyme which is activated by CoASAc or fructose 1, 6-diphosphate and inhibited by L-aspartate. The effect of CoASAc and aspartate on kinetic properties of the enxyme was extensively studied. 1. Among the three reaction components (PEP, HCO3− and Mg2+), PEP was the only component, the kinetic properties of which were remarkably affected by the effectors. 2. CoASAc increased the affinity of the emzyme for PEP and increased Vmax attainable at the infinite concentration of PEP. On the other hand, aspartate decreased the affinity for PEP but caused no effect on Vmax. Therefore, it became clear that the effect of CoASAc and aspartate is not just antipodal to one another, though apparently they are antagonistic. 3. The shape of PEP-saturation curve was not sigmoidal in the absence of the effector (Hill's coefficient, n=1.1), but, curiously, the sigmoidicity increased significantly with increasing concentrations of CoASAc in a range of concentrations of 0–0.05 mM (n=1.1–1.6). By further addition of CoASAc (0.1 mM), it decreased to 1.3. When aspartate (2.5 mM) was added to the reaction mixture which contained CoASAc (0.5 mM), the curve was again converted to remarkably sigmoidal one (n=2.5). 4. With increasing concentrations of PEP the affinity for CoASAc increased and the shape of CoASAc-saturation curve was converted from sigmoidal to hyperbolic one. The addition of aspartate decreased the affinity for CoASAc and increased the sigmoidicity of CoASAc-saturation curve. The quantitative investigation of the CoASAc concentrations requried for compensating the inhibition caused by various concentrations of aspartate, revealed that the order of aspartate action was apparently higher than that of CoASAc action. 5. The three-state model was presented for this enzyme to explain the kinetic features described in the present and previous reports (Biochim. Biophys. Acta, 139, 188 (1967)).