Abstract S100A4 (Mts1) is a Ca 2+-binding protein of the S100 family. This protein plays an important role in promoting tumor metastasis. In order to identify S100A4 interacting proteins, we have applied the yeast two-hybrid system as an in vivo approach. By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family. The non-covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mutational analysis revealed that replacement of Cys 76 and/or Cys 81 of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo.