Plasmodium species have the capacity to fix carbon dioxide during intracellular development. This process contributes to the pool of free amino acids and metabolites, which are the end products of glucose metabolism in the malaria parasite. A gene encoding phosphoenolpyruvate carboxykinase (PEPCK), an enzyme known to catalyze CO(2) fixation was identified in the genome of the human parasite Plasmodium falciparum by DNA microarray analysis experiments and was cloned and characterized. PfPEPCK is a 66.2 kDa, ATP-dependent enzyme which is closely related to PEPCK from plants and yeast but markedly different from the host enzyme human PEPCK. PfPEPCK transcript and active enzyme levels are upregulated in the transmissible and zygote stages of parasite development relative to the asexual blood stages. Elevated expression of PfPEPCK during the extracellular zygote phase of P. falciparum development within the microenvironment of the mosquito midgut may reflect a glucose-rare medium and suggests a possible switch in carbohydrate metabolism to a gluconeogenesis pathway.