High-resolution proton nuclear magnetic resonance spectroscopy and relaxation techniques have been used to investigate the interactions of sickle cell hemoglobin (Hb S) and human normal adult hemoglobin (Hb A) with p-bromobenzyl alcohol, L-phenylalanine, L-tryptophan, and L-valine. With the exception of valine, all these compounds inhibit the polymerization of deoxy-Hb S [Noguchi, C. T., & Schechter, A. N. (1978) Biochemistry 17, 5455)). Using transferred nuclear Overhauser effects among the proton resonances of the compound of interest and the corresponding longitudinal relaxation rates (T1(-1], we have shown that the binding of each of the compounds investigated to deoxy-Hb S is comparable to that to deoxy-Hb A. Intermolecular transferred nuclear Overhauser effects have been observed between proton resonances of the anti-sickling compounds and specific protons situated in the heme pockets of Hb. On the basis of these results, we suggest that one binding site, common to all compounds with anti-sickling activity, is at or near the heme pockets in the alpha and beta chains of both deoxy-HB S and deoxy-Hb A. The proton T1(-1) values of the histidyl residues situated over the surface of the hemoglobin molecule indicate that a second binding site is located at or near the beta 6 position, containing the mutation in Hb S (beta 6Glu----Val). The binding of the compounds investigated to the latter site induces conformational changes in the amino-terminal domains of the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)