We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K ؉ , and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system. Toxoplasma gondii is an obligate intracellular protozoan parasite of warm-blooded animals, including humans (1). Although normally asymptomatic, toxoplasmosis is a significant problem in pregnant women infected early during gestation, immunocompromised individuals, and livestock. This parasite is a member of the phylum Apicomplexa, which includes many other parasites such as Plasmodium species responsible for malaria. Glucose is thought to be the main source of energy for the rapidly multiplying forms of both Toxoplasma and Plasmodium, which use the Embden-Meyerhof pathway for glycolytic phosphorylation (2).Pyruvate kinase catalyzes the essentially irreversible transphosphorylation from phosphoenolpyruvate (PEP) 4 to ADP-producing pyruvate (3). In most mammals and bacteria, pyruvate kinase is allosterically regulated by fructose 1,6-diphosphate (4) and thus plays a regulatory role in glycolysis. The product pyruvate feeds into many metabolic pathways, placing pyruvate kinase at a crucial metabolic intersection. Many organisms express multiple pyruvate kinase isozymes with different kinetic properties. For example, Escherichia coli bears two isozymes, type I and II, both of which are homotropically activated by the substrate PEP. The type I isozyme is also activated heterotropically by fructose 1,6-diphosphate and is inhibited by ATP (5), whereas the type II isozyme is activated by AMP and mo...