Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with Zn-65-MT or (ZnCl2)-Zn-65 and the proteins receiving Zn-65 were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl Sulfate-PAGE (SDS PAGE), and detected by autoradiography. A unique Zn-65-binding band was observed from the Zn-65-MT-incubated, but not the (ZnCl2)-Zn-65-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction. (c) 2005 Elsevier Inc. All rights reserved.