The glyoxalase pathway ofLeishmania infantumwas kinetically character-ized as a trypanothione-dependent system. Using time course analysis based on parameter fitting with a genetic algorithm, kinetic parameters were estimated for both enzymes, with trypanothione derived substrates. A Kmof mmand aVof )1 Æmg )1 for glyoxalase I, and a Kmof mmand aVof )1 Æmg )1 for glyoxalase II, were obtained.