Microcalorimetry and equilibrium gel filtration were used to determine the thermodynamic functions ΔH°, ΔG° and ΔS° guiding the interaction of Ca²⁺ and Sr²⁺ with bovine α‐lactalbumin. Two methods of nearly complete metal removal from the protein gave identical results. The single Ca‐ and Sr‐binding site, which has moderate affinity for these ions (K_ca = 2.5 × 10⁶ M⁻¹ and K _Sr = 5.1 × 10⁵ M⁻¹), displays unusually large enthalpy changes of −118 kJ · mol⁻¹for Ca ²⁺ and −75 kJ·mol⁻¹ for Sr ²⁺. The concomitant reaction entropies equal −273 and −142 J·K⁻¹· mol ⁻¹, respectively.