Mitochondrial ATP synthase is responsive to changes in cytosolic calcium concentration, but the regulatory mechanisms are unclear. Here we identified a major 52 kDa calcium-binding protein in rat enamel cells as the mitochondrial ATP synthase F1-beta-subunit. The F1-beta-subunit behaved as a low affinity and moderate capacity calcium-binding protein during comparative 45Ca overlay analyses. Equivalent behavior was shown by the F1-beta-subunit from rat liver mitochondria, but not by the homologous F1-alpha-subunit, supporting the specificity of calcium binding. Evidence that the catalytic F1-beta-subunit binds calcium specifically introduces new mechanistic possibilities for regulating ATP synthase, and thereby coordinating ATP production with demand for ATP-fuelled calcium pump activity.