AbstractThe cationic dye auramine O forms a fluorescent complex with Ca2+‐liganded calmodulin. One moderately strong binding site is present, as well as one or more weaker sites. The binding site for auramine O is different from those for toluidinyl‐naphthalene sulfonate. The dependence of binding upon electrolyte concentration suggests a substantial electrostatic component of the free energy of binding. The splitting of the bond between residues 77 and 78 by trypsin digestion abolishes auramine O binding; the N‐ and C‐terminal half‐molecules have virtually no binding capacity. This suggests that the primary binding site is located near the midpoint of the connecting strand and includes elements of both half‐molecules. Thrombin digestion, which splits calmodulin between residues 106 and 107, also substantially reduces auramine O binding; this may be interpreted in terms of the stabilization of the structure of the connecting strand by interaction with residues within binding domain IV. The binding affinity at pH 5.0, where the helical organization of the connecting strand may be intact, is greater than at