We have cloned two gene (aldo-1 and aldo-2) encoding the glycolytic enzyme aldolase of the rodent malaria parasite Plasmodium berghei. The amino acid sequence of one gene product, ALDO-1, is virtually identical to P. falciparum aldolase whereas ALDO-2, the second gene product, is different and has 13% sequence diversity to ALDO-1. We expressed ALDO-2 as an active enzyme in Escherichia coli and compared the biochemical and kinetic properties to that of P. falciparum recombinant aldolase (ALDO-1 type). Based on the Km and Vmax constants for FMP and FBP, neither ALDO-1 nor ALDO-2 can be clearly assigned to any of the known mammalian isoenzyme classes. We demonstrate that expression of the two isoenzymes is developmentally regulated: specific antibody probes detect ALDO-1 in sporozoite stages of P. berghei and ALDO-2 is found in blood stage parasites.