The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca 2+-binding and target interaction properties. Here, our current understanding of the Ca 2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11–14 amino acid residues in the Ca 2+-binding loop are analyzed within the framework of the recently proposed two-step Ca 2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca 2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca 2+-binding loops in the two-EF-hand domain. This structure, named EFβ-scaffold, defines the position of the bound Ca 2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca 2+-binding loop. It is proposed that the nature of the first ligand of the Ca 2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca 2+-induced conformational change in the EF-hand proteins.