Chaperone proteins have been initially identified by their ability to confer cellular resistance to various stress conditions. However, molecular chaperones participate also in many constitutive cellular processes. Mitochondria contain several members of the major chaperone families that have important functions in maintaining mitochondrial function. The major Hsp70 of the mitochondrial matrix (mtHsp70) is essential for the translocation of cytosolic precursor proteins across the two mitochondrial membranes. MtHsp70 interacts with the preprotein in transit in an ATP-dependent reaction as it emerges from the translocation channel of the inner membrane. Together with two essential partner proteins, Tim44 and Mge1, mtHsp70 forms a membrane-associated import motor complex responsible for vectorial polypeptide movement and unfolding of preprotein domains. Folding of newly imported proteins in the matrix is assisted by the soluble chaperone system formed by mtHsp70 and its partner protein Mdj1. For certain substrate proteins, the protected folding environment that is offered by the large oligomeric Hsp60 complex facilitates further folding reactions. The mitochondrial Hsp70 Ssq1 is involved in the assembly of mitochondrial Fe/S clusters together with another member of the DnaJ family, Jac1. Chaperones of the Clp/Hsp100 family mediate the prevention of aggregation under stress conditions and eventually the degradation of mitochondrial proteins. Together, the chaperones of the mitochondrial matrix form a complex interdependent chaperone network that is essential for most reactions of mitochondrial protein biogenesis.