The antibiotic phosphomycin (1,2-epoxypropylphosphonic acid), an analog of phosphoenolpyruvate (PEP), behaved not as an inhibitor, but as an activator, of the enzyme phosphoenolpyruvate carboxylase (PEPC) from maize leaves. Multiple activation studies indicated that the analog binds to the Glc6P-allosteric site producing a more activated enzyme than Glc6P itself. Because of this, we used phosphomycin as a tool to further extend our understanding of the mechanisms of allosteric regulation of C4-PEPC. Initial velocity data from detailed kinetic studies, in which the concentrations of free and Mg-complexed PEP and phosphomycin were controlled, are consistent with: (1) the true activator is free phosphomycin, which competes with free PEP for the Glc6P-allosteric site; and (2) the Mg-phosphomycin complex caused inhibition by binding to the active site in competition with MgPEP. Therefore, although the Glc6P-allosteric site and the active site are able to bind the same ligands, they differ in the form of substrate and activator they bind. This important difference allows the full expression of the potential of activation and prevents inhibition by the activators, including the physiological ones, which are mostly uncomplexed at physiological free Mg2+ concentrations. At fixed low substrate concentrations, the saturation kinetics of the enzyme by phosphomycin showed positive cooperativity at pH 7.3 and 8.3, although at the latter pH, the kinetics of saturation by the substrate was hyperbolic. The cosolute glycerol greatly increased the affinity of the enzyme for phosphomycin and abolished the cooperativity in its binding, but did not eliminate the heterotropic effects of the activator. Therefore, the heterotropic and homotropic effects of the activator are not always coupled to the homotropic effects of the substrate, which argues against the two-state model previously proposed to explain the allosteric properties of maize-leaf PEPC.