The ‘EF-hand’ Ca²⁺-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix–loop–helix secondary structure as well as the ligands presented by the loop to bind the Ca²⁺ ion. The identity of these ligands is semi-conserved in the most common (the ‘canonical’) EF-hand; however, several non-canonical EF-hands exist that bind Ca²⁺ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca²⁺ signal required to reach protein saturation. The conformational effects of Ca²⁺ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca²⁺, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca²⁺ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca²⁺: selectivity over Mg²⁺ (a cation with very similar chemical properties to Ca²⁺ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca²⁺ with a wide range of affinities.