Two site-directed mutants of human promyeloperoxidase, MPO(His416----Ala) and MPO(His502----Ala), have been expressed in Chinese hamster ovary cells and purified. Overall purification yields and apparent molecular masses of the mutant proteins were similar to those of the wild-type enzyme. Both mutant species were analyzed spectroscopically to check the presence of the hemic iron in the proteins and were assayed for peroxidase activity. The data show that substitution of His502 leads to the loss, or to an inappropriate configuration, of the heme together with the loss of activity, suggesting that this residue could be the proximal His involved in the binding to the iron centers. On the other hand, substitution of His416 by alanine had no effect on either of the studied parameters.