Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D synthase, and granulin precursor were identified as proteins interacting with the N-terminal half of mammalian Type III hexokinase (HKIII) in the yeast two-hybrid method. These interactions were confirmed by in vitro binding studies. All five of these proteins, and their mRNAs, were present in PC12 cells, as shown by immunoblotting and RT-PCR, respectively. All were coimmunoprecipitated from PC12 extracts with an antibody against HKIII, but not with anti-Type I hexokinase. Moreover, all of these proteins were coimmunoprecipitated using antileptin as precipitating antibody, indicating the existence of a macromolecular complex including these five proteins and HKIII. Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) reporter constructs gave a diffuse intracellular fluorescence. Cotransfection with leptin or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion protein, at intracellular sites coincident with localization of leptin-GFP or Miz-1-GFP reporter constructs.