Pyruvate dehydrogenase phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by Ca2+-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca2+ was evaluated in activity and L2 binding studies. An increase in the Mg2+ concentration decreased the Ca2+ concentration required for half-maximal activation of PDP1 from 3 to 1 μM, but this parameter was unchanged at 3 μM with PDP1c. Near 1 μM Ca2+, tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg2+. With just Ca2+ included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c·Ca2+·L2 complex is supported by micromolar Ca2+ concentrations and becomes sensitive to the Mg2+ level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 ± 2.5 μM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K d of ∼0.3 μM. Isothermal titration calorimetry and 45Ca2+ binding studies failed to detect binding of Ca2+ (<100 μM) to L2 or PDP1c, alone, but readily detected binding to L2 and PDP1c. Therefore, both proteins are required for formation of a complex with tightly held Ca2+, and complex formation hinders the tendency of PDP1c to form a dimer.