Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of P_i starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104‐kDa PEPC subunit. To further assess the influence of P_i deprivation on PEPC, the enzyme was purified from P_i‐sufficient (+P_i) and P_i‐starved (–P_i) cells to electrophoretic homogeneity and final specific activities of 37–40 µmol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +P_i and –P_i PEPCs are both homotetramers composed of an identical 104‐kDa subunit. Respective pH–activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to l‐malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +P_i and –P_i cells exists mainly in its dephosphorylated (l‐malate sensitive) form. Thus, up‐regulation of PEPC activity in –P_i cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +P_i cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6‐phosphate and inhibition by l‐malate, l‐aspartate, l‐glutamate, dl‐isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. l‐Aspartate and l‐glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.