To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, IIalpha-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.