The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23(lMs)) and the interaction of presequence peptides with Tim23(lMs) in presence of Ca2+ ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23(lMs). The order of the binding strength is Ca2+, Mg2+, Cu2+, Mn2+, and Co2+ (from strong to weak). Moreover, the interaction of presequence peptides with Tim23lMs is weakened in presence of Ca2+ ion, which implicates that Ca2+ ion may play an important role in the protein import by TIM23 complex. (C) 2016 Elsevier Inc. All rights reserved.