Abstract: Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23(SORT)). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23(SORT) and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import.