N-Myc downregulated gene (NDRG) 1 is markedly induced during in vitro maturation of mouse immature bone marrow-derived mast cells (BMMCs) into a mature connective tissue mast cell (CTMC)-like phenotype. However, cellular function of this unique cytosolic protein is currently obscure. In this study, we sought potential NDRG1-binding proteins using yeast two-hybrid analysis and found that NDRG1 is capable of binding to heat-shock cognate protein 70 (Hsc70) both in vitro and in mast cells. The expression of Hsc70 was markedly elevated during the in vitro maturation of BMMCs into CTMC-like cells in accordance with the increased expression of NDRG1. Deletion of the C-terminal hydrophilic tandem repeats from NDRG1 facilitated the interaction with Hsc70 in vitro. Interaction between NDRG1 and Hsc70 was constitutive in mast cells and was not altered following cell activation. Although NDRG1 undergoes phosphorylation (accompanying paper), the binding of NDRG1 to Hsc70 was not affected by this event. Interestingly, the NDRG1-Hsc70 complex transiently appeared in the nuclear fraction of activated mast cells.