The mutant hemoglobin S (HbS) previously was reported to undergo accelerated autooxidation during incubation in vitro. However, subsequent observations have raised the possibility that this might be explained by adventitious association of molecular iron with HbS, rather than reflecting an inherent property of HbS. Using purified HbA and HbS obtained from genotypic HbAS donors, we found that the observed oxidation rate of HbS, but not of HbA, is indeed exaggerated by adventitious iron. This result suggests a preferential partitioning of molecular iron to HbS over HbA, which was further supported by experimentation. However, after elimination of this effect, there still remains a significant increase in inherent autooxidation rate for HbS. Physiologic oxidants (superoxide, peroxide, hydroxyl radical) and various Fe(III) chelates all stimulate oxidation of oxyHb, but they do so equivalently for HbA and HbS. Nevertheless, these mechanisms also would contribute to excessive biologic oxidation of HbS because the cytoplasm of sickle red blood cells, unlike that of normal cells, would be exposed to abnormal amounts of oxidants and low-molecular-weight iron compounds.