Two isoforms of phosphoenolpyruvate carboxylase (PEPC) with very different regulatory properties were partially purified from the green alga Selenastrum minutum. They were designated PEPC(1) and PEPC(2). PEPC(1) showed sigmoidal kinetics with respect to phosphoenolpyruvate (PEP) whereas PEPC(2) exhibited a typical Michaelis-Menten response. The S(0.5)(PEP) of PEPC(1) was 2.23 millimolar. This was fourfold greater than the S(0.5)(PEP) of PEPC(2), which was 0.57 millimolar. PEPC(1) was activated more than fourfold by 2.0 millimolar glutamine and sixfold by 2.0 millimolar dihydroxyacetone phosphate (DHAP) at a subsaturating PEP concentration of 0.625 millimolar. In contrast, PEPC(2) showed only 8% and 52% activation by glutamine and DHAP, respectively. The effects of glutamine and DHAP were additive. PEPC(1) was more sensitive to inhibition by glutamate, 2-oxoglutarate, and aspartate than PEPC(2). Both isoforms were equally inhibited by malate. All of these metabolites affected only the S(0.5)(PEP) not the V(max). The regulatory properties of S. minutum PEPC in vitro are discussed in terms of (a) increased rates of dark carbon fixation (shown to be catalyzed predominantly by PEPC) and (b) changes in metabolite levels in vivo during enhanced NH(4+) assimilation. Finally, a model is proposed for the regulation of PEPC in vivo in relation to its role in replenishing tricarboxylic acid cycle intermediates consumed in NH(4+) assimilation.