Glyoxalase I (GloI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. Studies on GloI from diverse organisms such as man, bacteria, yeast, and different parasites show striking differences among these potentially isofunctional enzymes as far as metal content and the number of active sites per subunit are concerned. So far, it is not known whether this structural variability is linked to catalytic or regulatory features in vivo. Here we show that recombinant GloI from the malaria parasite Plasmodium falciparum has a high- and a low-affinity binding site for the diastereomeric hemithioacetals formed by addition of glutathione to methylglyoxal. Both active sites of the monomeric enzyme are functional and have similar k(cat)(app) values. Proteolytic susceptibility studies and detailed analyses of the steady-state kinetics of active-site mutants suggest that both reaction centers can adopt two discrete conformations and are allosterically coupled. As a result of the positive homotropic allosteric coupling, P. falciparum GloI has an increased affinity at low substrate concentrations and an increased activity at higher substrate concentrations. This could also be the case for GloI from yeast and other organisms. Potential physiologically relevant differences between monomeric GloI and homodimeric GloI are discussed. Our results provide a strong basis for drug development strategies and significantly enhance our understanding of GloI kinetics and structure-function relationships. Furthermore, they extend the current knowledge on allosteric regulation of monomeric proteins in general.