Erythrocyte pyruvate kinase (RPK) is a key glycolytic enzyme, essential for cellular energy supply. Disfunction of RPK lead to hemolytic non spherocytic anemia, an hereditary disease transmitted as a autosomic recessive trait. To date more than 150 pathological mutations have been identified, but the difficulty to isolate mutant enzymes from patient's blood prevented an accurate characterization. In order to delucidate the molecular bases of the erythroenzymopathy, and to define the functional role of the mutated amino acids, we have performed a structural and functional analysis of the wild type RPK in recombinant form and of ten pathological variants obtained with site directed mutagenesis. The mutations selected target into the domain interfaces, the hydrophobic core and the allosteric site.