Calmodulin (CaM) is a major Ca 2 messenger which, upon Ca 2 activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca 2 ion concentration suggests that CaM activation may be modulated by low-affinity Ca 2 chelators. The effect on CaM structure and function of citrate ion, a Ca 2 chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg 106 -His 107 . Owing to the tight proximity of this site to the third Ca 2binding loop of CaM, binding of citrate is presumably translated into changes in Ca 2 binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein.