To demonstrate and characterize red cell inclusions in 101 persons with Hb S or Hb C disorders three methods were used: (1) examination of unstained blood smears by dark field microscopy (DFM), (2) examination of blood smears after acid elution and staining (AE), and (3) measurement of membrane-associated denatured haemoglobin (MADH) in ghosts. The control group had inclusions in less than 5% of red cells by DFM and AE and the mean percentage of MADH per total cellular Hb was 0.030+/-0.016%. The highest percentages of red cells with inclusions and of MADH were present in clinically severe haemoglobin disorders, e.g. homozygous sickle cell disease (Hb SS) with less than 10% Hb F and Hb SOArab, with successively lower percentages in moderate to severe disorders, e.g. Hb SS-alpha thalassaemia, Hb-S-beta0 thalassaemia, Hb SC disease, and Hb SS with more than 10% Hb F, indicating agreement in results by three methods. In asymptomatic or mild disorders, e.g. Hb-S-beta+ thalassaemia, Hb CC, Hb AC and Hb AS, the results by AE and measurements of MADH were the same or similar to those in controls, while those by DFM were different. Of 56 patients with Hb SS or Hb SC, the group with functional asplenia had higher percentages of MADH and of red cells with inclusions than those with functioning spleens. Our study suggests that inclusions in sickling disorders may be due to denatured Hb S, with AE being the more accurate method for visualizing these inclusions, as results by this method correlate better with the amount of MADH than those by DFM.