Evidence is presented for the existence of slowly exchanging Ca2(+)-binding sites in troponin C (CTNC) of cardiac and slow twitch skeletal muscles. These sites were revealed in the course of experiments aimed at measuring the Ca2(+)-binding properties of TNC in the myofilament lattice. 45Ca bound to chemically skinned muscle fibers or myofibrils of cardiac and soleus muscles was eluted by EGTA in a two-exponential timecourse with a slow phase of a rate constant of about 2 x 10(-4)/s. The slow phase was not found in skinned fiber or myofibrils of psoas, a fast skeletal muscle. However, skinned psoas fibers in which the native TNC was replaced by CTNC exhibited the slow 45Ca elution characteristic of soleus and cardiac preparations, indicating that the slowly-exchanging sites are located in CTNC. These sites are tentatively identified as the Ca2(+)-Mg2+ sites of CTNC on the basis that the slow phase was observed under conditions known to restrict Ca2+ binding to the Ca2(+)-Mg2+ sites.