The EF-hand calcium-binding loop III from calmodulin was inserted with glycine linkers into the scaffold protein CD2.D1 at three locations to study site-specific calcium binding properties of EF-hand motifs. After insertion, the host protein retains its native structure and forms a 1:1 metal-protein complex for calcium and its analog, lanthanum. Tyrosine-sensitized Tb3+ energy transfer exhibits metal binding and La3+ and Ca2+ compete for the metal binding site. The grafted EF-loop III in different environments has similar La3+ binding affinities, suggesting that it is largely solvated and functions independently from the host protein.