The soluble pool of α hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, β A hemoglobin chain labeled with [³H]N-ethylmaleimide. This pool of soluble α chains was 0.067 ± 0.017% of hemoglobin in blood of normal adult, 0.11 ± 0.03% in heterozygous β thalassemia and ranged from 0.26 to 1.30% in homozygous β thalassemia intermedia. This elevated pool of soluble α chains observed in human β thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble α chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In β thalassemia intermedia the amount of insoluble α chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [³H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.