We have cloned a cDNA encoding Toxoplasma gondii pyruvate kinase and obtained the full-length recombinant enzyme with a calculated molecular mass of 57.5 kDa. The predicted amino acid sequence of T. gondii pyruvate kinase exhibited a highest identity (63%) to that of Eimeria tenella pyruvate kinase and a lower identity of less than 25% to the pyruvate kinases from other organisms. Southern blot analysis indicated that the pyruvate kinase gene existed as a single copy in the T. gondii tachyzoite. The active recombinant enzyme contained four subunits and produced a strongly sigmoid saturation curve with phosphoenolpyruvate as the variable substrate. Fructose 1,6-diphosphate, a general activating factor of pyruvate kinase in most species, did not affect the enzyme activity. However, glucose 6-phosphate radically activated the enzyme. Fructose 2,6-diphosphate suppressed the reaction velocity at a higher concentration of phosphoenolpyruvate. These properties indicate that pyruvate kinase activity in T. gondii is regulated by unusual phosphorylated sugars.