Mitochondria import the vast majority of their proteins from the cytosol. The mitochondrial import motor of the TIM23 translocase drives the translocation of precursor proteins across the outer and inner membrane in an ATP-dependent reaction. Tim44 at the inner face of the translocation pore recruits the chaperone mtHsp70, which binds the incoming precursor protein. This reaction is assisted by the cochaperones Tim14 and Mge1. We have identified a novel essential cochaperone, Tim16. It is related to J-domain proteins and forms a stable subcomplex with the J protein Tim14. Depletion of Tim16 has a marked effect on protein import into the mitochondrial matrix, impairs the interaction of Tim14 with the TIM23 complex and leads to severe structural changes of the import motor. In conclusion, Tim16 is a constituent of the TIM23 preprotein translocase, where it exerts crucial functions in the import motor.